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Microenvironment and Immunology

HIF-2a Enhances b-Catenin/TCF-Driven Transcription byInteracting with b-Catenin

Hyunsung Choi1, Yang-Sook Chun2, Tae-You Kim3, and Jong-Wan Park1

AbstractThe tumor-promoting factors b-catenin and hypoxia-inducible factor (HIF) are often found to be coactivated

in rapidly growing tumors. Recently, it was shown that HIF-1a negatively regulates Wnt/b-catenin signaling bysequestering b-catenin from b-catenin/T-cell factor (TCF). However, no investigation has been undertaken onthe involvement of HIF-2a in b-catenin regulation. In this study, it was found that, like HIF-1a, HIF-2a interactswith b-catenin, but at a different site. Furthermore, HIF-2a was found to assemble with b-catenin/TCF andfacilitate gene transcription. Mutational analyses revealed that transactivation domains of HIF-2a promote p300coactivator recruitment by b-catenin. Furthermore, HIF-2a and b-catenin were found to associate in the nucleiof 786-0 renal cell carcinoma cells, and HIF-2a was found to be required for b-catenin activation in these cellsand for their proliferation. These results suggest that this interaction contributes to the unrestrained growth oftumor cells containing coactivated HIF-2a and b-catenin. Interestingly, these actions of HIF-2a oppose those ofHIF-1a on b-catenin and cell growth, and this suggests that HIF-1a/HIF-2a balance may importantly determinecell growth when hypoxia and Wnt stimulation coexist. Cancer Res; 70(24); 10101–11. �2010 AACR.

Introduction

Hypoxia commonly develops during tumor growth in can-cer and is associated with a poor prognosis (1). Tumoradaptation to hypoxia is mainly mediated by 2 transcriptionfactors, that is, the hypoxia-inducible factors (HIFs) HIF-1 andHIF-2. HIF-1 is composed of HIF-1a and aryl hydrocarbonreceptor nuclear translocator (ARNT/HIF-1b), and HIF-2 ofARNT and HIF-2a (2, 3). The a subunits (HIF-as), which areregulated by oxygen tension, function as prime transactivatingfactors. Under normoxic conditions, HIF-as are hydroxylatedat their proline and asparagine residues by HIF-prolyl hydro-xylases (PHD1–3) and factor inhibiting HIF (FIH), respectively(4, 5). Proline hydroxylation then induces Von Hippel–Lindauprotein (pVHL)-mediated ubiquitination and the proteasomaldegradation of HIF-as, and asparagine hydroxylation func-tionally inhibits HIF-as by blocking the recruitment of p300/CBP coactivators. Under hypoxic conditions, however, HIF-asare stabilized and activated because both hydroxylations arelimited.

Given the structural similarities of HIF-1a and HIF-2a, itwas believed that they exhibit redundancy in terms ofcellular response to hypoxia. However, growing evidencesuggests that HIF-1a and HIF-2a express different sets ofgenes (6). In addition, they play differential roles in deter-mining cell fate under hypoxia. HIF-1a induces cell cyclearrest during hypoxia, which is attributed to its interactionwith factors that promote cell cycle. For example, HIF-1aantagonizes c-Myc by sequestering MAX, which leads to thedownregulation of c-Myc–targeted proliferative genes and tothe upregulation of p21WAF1/Cip1 (7). In contrast, HIF-2apromotes the growth of clear cell renal cell carcinoma(ccRCC), lung carcinoma, and neuroblastoma cells. HIF-2a also interacts with MAX, but stabilizes and activatesthe c-Myc/MAX complex (8). Given that c-Myc potentlydrives unrestrained cell proliferation, the counterbalancingeffects of HIF-1a and HIF-2a on c-Myc regulation maycritically determine whether cancer cells are proliferativeor quiescent during hypoxia.

b-Catenin, the prime effecter of canonical Wnt signaling,expresses numerous genes responsible for cell proliferation,differentiation, and survival. When Wnt ligands bind toFrizzled receptor, disheveled (Dsh) inhibits GSK3b, and inturn blocks the phosphorylation and degradation of b-catenin.Stabilized b-catenin dimerizes with TCF/LEF factor in thenucleus, and transactivates proliferative genes, such as, c-Mycand cyclin D1 (9). In many tumors, b-catenin has been foundto be aberrantly activated and to act as a tumor-promotingfactor (10). Kaidi et al. (11) proposed that the hypoxia-inducedgrowth arrest is attributed to HIF-1a inactivation of b-catenin.HIF-1a dissociates b-catenin/TCF4 by directly sequesteringb-catenin in hypoxia. Interestingly, this scenario is similar tothemechanism by which HIF-1a inactivates c-Myc, namely, by

Authors' Affiliations: Departments of 1Pharmacology, 2Physiology, and3Internal Medicine, Ischemic/Hypoxic Disease Institute, Seoul NationalUniversity College of Medicine, Seoul, Republic of Korea

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Jong-Wan Park, Department of Pharmacology,Seoul National University College of Medicine, 28 Yongon-dong,Chongno-gu, Seoul 110–799, Republic of Korea. Phone: 82 2 740 8289;Fax: 82 2 745 7996; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-0505

�2010 American Association for Cancer Research.

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dissociating heterodimeric factors by sequestering one of itssubunits.

Given that the c-Myc regulatory roles of HIF-2a and HIF-1aare opposed, we sought to investigate whether their roles arealso opposed during b-catenin regulation. Therefore, wetested the possibility that HIF-2a associates with and acti-vates b-catenin/TCF. Moreover, in 786-0 ccRCC, HIF-2a wasfound to naturally associate with b-catenin and to promotecell proliferation. This study provides another mechanismwhereby HIF-as determine the growth status of tumor cellsduring hypoxia.

Materials and Methods

MaterialsLithium chloride and tag peptides (Myc, hemagglutinin

(HA), and FLAG) were purchased from Sigma-Aldrich.Anti–HIF-1a antiserum was generated in rabbits, as describedpreviously (12). Anti–HIF-2a (C terminus) antiserum wasobtained from Novus Biologicals; anti-HA tag, anti-Myc tag,and anti-FLAG tag from Sigma-Aldrich; anti-p21 from BDBioscience; anti-Axin2 from Cell Signaling Technology; fluor-escein isothiocyanate (FITC)-conjugated anti-bromodeoxyur-idine (BrdU) from BD Pharmingen. Other primary andhorseradish peroxidase (HRP)-conjugated secondary antibo-dies were purchased from Santa Cruz Biotechnology.

Cell cultureHEK293T embryonic kidney and 786-0 RCC cell lines were

authenticated by the supplier (American Type Culture Collec-tion) based on growth, morphology, and isoenzymology.HEK293T and 786-0 cells were cultured in DMEM and RPMI1640 medium, respectively, with 10% of heat-inactivated fetalbovine serum. On arrival, cells were cultured for 5 passages,and then frozen in multiple vials. After thawing, cells werekept in culture for less than 15 passages. No authentication,other than cell growth and morphology, of the cell lines wasperformed during experiments.

siRNAs, plasmids, and transfectionSmall interfering RNAs (siRNAs) were provided from Inte-

grated DNA Technologies. Plasmids of HIF-1a, HIF-2a, b-cate-nin, and TCF4 were constructed by RT-PCR and blunt-endligation, as previously described (13). For transient genesilencing or protein expression, approximately 40% confluentcells were transfected with siRNAs or plasmids using calciumphosphate, Lipofectamine (Invitrogen), or Nucleofector elec-troporation (Lonza). Cells were allowed to stabilize for 48hours before being used in the experiments.

Reporter assayTOPflash reporter was obtained from Millipore and VEGF-

promoter reporter was a kind gift from Dr. Eric Huang(University of Utah). FOPflash reporter lacking b-catenin/TCF4-binding site and pCBG99 SV40-promoter reporter wereused as controls. Cells were cotransfected with 0.5 to 2 mgeach of reporter and cytomegalovirus (CMV)–b-gal plasmid.Luciferase activity was measured using Lumat-LB960

luminometer (Berthold), and divided by b-gal activity tonormalize transfection efficiency.

Immunoblotting and immunoprecipitationProteins were separated on 8% to 10% SDS-polyacrylamide

gel, and transferred to Immobilon-P membranes (Millipore).Membranes were blocked with 5% milk in TBS for 1 hour andincubated overnight at 4�C with primary antibodies (1:1,000),and sequentially with HRP-conjugated secondary antibodies(1:5,000). The immune complexes were visualized usingenhanced chemiluminescence–plus (Invitrogen). For immu-noprecipitation, cell lysates were incubated with anti-Myc/HA/FLAG beads for 2 hours, and then eluted with Myc/HA/FLAG peptides.

Cell cycle and proliferation analysesCells were harvested, fixed in 75% ethanol, and labeled with

propodium iodide (0.05 mg/mL) for 30 minutes. DNA-incor-porated propodium iodide was detected at 488/650 nm usingFACStar flow cytometer (BD Bioscience). To analyze cellproliferation, cells were treated with 10 mol/L BrdU for 30minutes, fixed, permeabilized, treated with DNase, and incu-bated with FITC-conjugated anti-BrdU. Total DNAs werestained with 7-AAD. FITC and 7-AAD were excited at488 nm and detected at 515 to 565 nm and 630 to 660 nm,respectively.

Immunofluorescent stainingCells were fixed with 3.7% paraformaldehyde, blocked with

3% bovine serum albumin, and incubated with anti–HIF-2a(1:300) and anti–b-catenin (1:300) overnight at 4�C. Cells wereincubated with biotinylated secondary antibodies (1:1,000)and reacted with ALEXA fluor-488/green conjugated strepta-vidine (1:1,000) or ALEXA fluor-594/red-conjugated streptavi-dine (1:1,000) for 1 hour. All nuclei were counterstained with0.1 mg/mL of 40, 6 diamidino 2 phenylindole (DAPI). Fluores-cence images were observed using IX71 microscope (Olym-pus) and captured by UIS 2 Olympus camera.

Preparation of nuclear extractAfter spun down, cell pellets were resuspended in 0.6% NP-

40, centrifuged, and the nuclear pellets were resuspended in ahigh osmotic solution with 400 mmol/L NaCl and 5% glycerol.After launching on shaking incubator at 4�C for 30 minutes,the sample was centrifuged at 15,000�g for 5 minutes at 4�Cand the supernatant was collected as the nuclear fraction.

Chromatin immunoprecipitation and qPCRCells were treated with 10% formaldehyde for 10 minutes

and then with 125 mmol/L glycine for 5 minutes. Cells werelysed, sonicated, and chromatins were immunoprecipitatedwith nonimmunized (IgG), anti–b-catenin, or anti–HIF-2aserum at 4�C overnight. Precipitated DNAs were amplifiedby qPCR (94�C, 52�C, and 70�C) and quantified by calculatingCt (threshold cycle) values between experimental and control(IgG) samples. The qPCR was carried out using DyNAmo SYBRGreen Kit (Finnzymes) and 7900HT real-time PCR (AppliedBiosystems). Coprecipitated TBE1 (�1,104 to �920 from the

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Cancer Res; 70(24) December 15, 2010 Cancer Research10102



c-Myc transcription site) and TBE2 (�604 to �358) segmentswere amplified using specific primer sets.

Statistical analysisAll data were analyzed using Microsoft Excel 2003, and

results are expressed as mean and SD. Data were statisti-cally analyzed using Mann–Whitney U Test, and differenceswere considered significant when P < 0.05 in 2-sided sta-tistics.

Other materials and methodsThey are described in the supplemental information.

Results

The interaction between HIF-2a and b-cateninTo examine the interactions among HIF-as, b-catenin,

and TCF4, HA-b-catenin or Myc-TCF4 was coexpressedwith HIF-1a or HIF-2a in HEK293T cells, and immunopreci-pitated with anti-HA or anti-Myc antibody. Ectopic b-catenincoprecipitated with HIF-1a and HIF-2a (Fig. 1A), but TCF4did not (Fig. 1B). These results indicate that both HIF-as areable to associate with b-catenin. To be functional, b-cateninand HIF-2a should bind with TCF/LEF and ARNT, respec-tively (3, 9). Therefore, we next addressed whether thesefactors assemble. We coexpressed HA-b-catenin, Myc-TCF4,HIF-2a, and ARNT in HEK293T and performed 2-stepimmunoprecipitations; b-catenin, TCF4, and HIF-2a,but not ARNT, associated all together (Fig. 1C). Next, weperformed Chromatin immunoprecipitation for the c-Mycgene; a b-catenin/TCF target. Of 2 TCF-binding elements(TBEs; ref. 14), TBE2 associated with b-catenin and HIF-2a,but TBE1 did not (Fig. 1D). Moreover, HIF-2a substantiallyincreased the b-catenin binding to TBE2. These results indi-cate that HIF-2a associates with b-catenin/TCF4 and parti-cipates in the b-catenin/TCF-driven transcription.

Sites of interaction in HIF-2a and b-cateninGiven that both HIF-as interact with b-catenin, we tested

the possibility that they compete with each other for b-cate-nin binding. Despite increasing HIF-2a expression, theb-catenin–HIF-1a interaction was unaffected (Fig. 2A,top). Likewise, the b-catenin–HIF-2a interaction was unaf-fected when HIF-1a was overexpressed (Fig. 2A, bottom).These findings indicate that HIF-1a and HIF-2a targetdifferent sites of b-catenin. We next tried to identify theirinteracting sites. In its central region, b-catenin possesses alarge armadillo repeat (AR) domain composed of 12 ARs,which provide a common binding site for TCF, ICAT, E-cadherin, and APC. Because the AR domain forms a rigidscaffold, protein binding occurs exclusively at this site (15).That is, when b-catenin (ARs 3–10) was occupied by TCF4,the central region of b-catenin may not bind to otherproteins any more. Therefore, we sought to determinewhether HIF-as interacts with the N terminus (includingARs 1–3) or C terminus (including ARs 10–12) of b-catenin.HIF-1a coprecipitated with the C terminus (aa. 530–781),which is consistent with a previous report (11), but HIF-2a

with the N terminus (aa. 1–259; Fig. 2B, left). We nextexamined whether the N-terminal end (aa. 1–140) lackingARs 1–3 binds to b-catenin, using the FLAG/SBP vector,which enlarges the short peptide to the size detectable byimmunoblotting. As a consequence, b-catenin is likely tointeract with HIF-2a through ARs 1–3 (Fig. 2B, right). Thus,because HIF-1a and HIF-2a interact at different sites, theymight not compete with each other for b-catenin binding.Then, do both HIF-1a and HIF-2a simultaneously bind tob-catenin? To check this possibility, we coexpressed theseproteins and performed coimmunoprecipitation analysis.HIF-1a and HIF-2a were coprecipitated in the presenceof b-catenin, whereas they were not in the absence ofb-catenin (Supplementary Fig. S1), suggesting the cobindingof the 2 HIF-as to b-catenin. On the contrary, we also tried toidentify the HIF-2a site for b-catenin binding. Immunopre-cipitation analyses showed that the HIF-2a N terminus (aa.1–395) is responsible for binding b-catenin (Fig. 2C). Tofurther narrow down the binding site, we deleted HIF-2aserially from N terminus. The HIF-2a mutant lacking aa.1–67 failed to interact with b-catenin (Fig. 2D), suggestingthat the N-terminal end (including bHLH) of HIF-2a parti-cipates in its binding with b-catenin. The binding sites ofb-catenin and its partners are summarized in Supplemen-tary Figure S2.

HIF-2a enhances the transcriptional activity ofb-catenin

HIF-1a functions to inhibit canonical Wnt signaling byinteracting with b-catenin (11). Then, what is the functionalconsequence of b-catenin–HIF-2a interaction? After treatingHEK293T cells with LiCl for 16 hours to activate b-catenin, weanalyzed b-catenin/TCF activity, using TOPflash reporter.HIF-2a expression significantly augmented the reporter activ-ity, whereas HIF-1a attenuated it (Fig. 3A, left). However,FOPflash and SV40-promoter reporters showed no responseto LiCl or HIF-1/2a overexpression, supporting the notion thatHIF-as act specifically on b-catenin/TCF. VEGF-promoterreporter analysis verified the functionalities of ectopic HIF-as (Fig. 3A, right). Even when cells were treated with LiCl forjust 4 hours, the b-catenin/TCF activity was significantlyenhanced by HIF-2a (Supplementary Fig. S3). To analyzethe activity of b-catenin in more detail, we coexpressedHA-catenin and TOPflash reporter in HEK293T. Reporteractivity was enhanced by HA-catenin, and the activity wasdiminished or enhanced by HIF-1a or HIF-2a, respectively(Fig. 3B). Moreover, the HIF-2a d67 mutant lacking theb-catenin-binding domain failed to increase the reporteractivity, further supporting that HIF-2a activates b-catenin/TCF by binding b-catenin. When HIF-2a fragments wereexpressed, the full-length HIF-2a alone activated b-catenin(Fig. 3C). This suggests that intact HIF-2a is required for thetranscriptional enhancement of b-catenin. To confirm thepositive role of HIF-2a in b-catenin activation, we analyzedthe expressions of b-catenin target proteins, Axin2 and c-Myc.HIF-2a significantly augmented the LiCl-stimulated expres-sion of both proteins, but the d67 mutant did not (Fig. 3D;Supplementary Fig. S4).

b-Catenin Activation by HIF-2a

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HIF-2a activates b-catenin by recruiting p300 throughits transactivation domains

To understand how HIF-2a activates b-catenin, we firstexamined whether HIF-2a promotes the nuclear translocationof b-catenin. After LiCl treatment, b-catenin increased innuclear fractions, but this was unaffected by HIF-2a

(Fig. 4A). In addition, we examined whether HIF-2a stabilizesb-catenin/TCF4 complex, but immunoprecipitation analysesrevealed that the coprecipitation of b-catenin and TCF4 wasnot increased by HIF-2a (Fig. 4B). We next tested the possi-bility that HIF-2a enhances b-catenin/TCF4-drived transcrip-tion through its transactivation domains (TAD). HIF-2a

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B

Figure 1. Interaction between HIF-2a and b-catenin. A, HIF-2a associates with b-catenin. HEK293T cells were cotransfected with HA-b-cateninand HIF-1/2a and then immuprecipitation was performed. Each plasmid was used at an equal dose of 4 mg per 100-mm dish. B, HIF-2a does not directlybind TCF-4. Myc-TCF-4 and HIF-1/2a were coexpressed in HEK293T cells. Immunoprecipitation was performed with anti-Myc and immunoblottingwas done with HIF-1/2a antibody. C, HIF-2a, not ARNT, is included in the b-catenin/TCF-4 complex. HEK293T cells were cotransfected with indicatedplasmids and further incubated for 48 hours. Double immunoprecipitations were done as illustrated in the left panel. HA-b-catenin/Myc-TCF-4complex was purified by sequential immunoprecipitations using anti-HA and anti-Myc, and coprecipitated proteins were identified by immunoblotting.D, HIF-2a targets the c-Myc gene. HEK293T cells were transfected with plasmids indicated. Chromatin-immunoprecipitation (ChIP) was performedusing nonimmunized serum (IgG), anti–b-catenin, or anti–HIF-2a, and then the coprecipitated DNAs were analyzed by qPCR. TBE-1 and -2 segments ofthe c-Myc gene were amplified with specific primers. *, P < 0.05 between 2 groups.

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harbors 2 TADs at its C-terminal part, namely, N-TAD at aa.530–652 and C-TAD at aa. 828–870 (16). To examine therequirement of HIF-2a TADs in b-catenin/TCF4 activation,we coexpressed TOPflash reporter with HIF-2a mutants lack-ing N-TAD, C-TAD, or lacking both TADs, which are desig-nated dN-TAD, dC-TAD, and dN/C-TAD, respectively (Fig. 4C,

bottom). Compared with full-length HIF-2a, dN-TAD and dC-TAD were significantly less effective at stimulating b-catenin,and dN/C-TAD had no effect (Fig. 4C, top). This findingindicates that both TADs of HIF-2a are involved in b-cateninactivation. To further investigate the role of HIF-2a in b-cate-nin activation, we examined the possible involvement of p300

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B

Figure 2. HIF-1a and HIF-2a interact with different domains of b-catenin. A, noncompetitive interaction between HIF-1a and HIF-2a for binding b-catenin.pHA-b-catenin (4 mg) was cotransfected into HEK293T cells with pHIF-1a (4 mg) and increasing amount (2, 4, 6 mg) of pHIF-2a (top) or with pHIF-2a(4 mg) with increasing amount (2, 4, 6 mg) of pHIF-1a (bottom). Protein interactions were analyzed by immunoprecipitation–immunoblotting. B, HIF-asinteracting domains of b-catenin. Myc-tagged N/C termini or FLAG/SBP-tagged N termini of b-catenin were coexpressed with HIF-1/2a in HEK293T cells, andimmunoprecipitated proteins were identified by immunoblotting. Each protein amount was estimated using the ImageJ program (NIH), and thefraction of precipitation to input was calculated (% Input). C, N terminus of HIF-2a interacts with b-catenin. Each FLAG-tagged HIF-2a fragment wascoexpressed with HA–b-catenin in HEK293T cells. After being immunoprecipitated with anti-HA, proteins were analyzed using anti-FLAG. D, the N-terminalend of HIF-2a is required for its binding to b-catenin. FLAG-tagged HIF-2a mutants derived from serial deletions were coexpressed with HA-b-catenin inHEK293T cells. Coimmunoprecipitation was done with anti-HA and anti–HIF-2a. *, nonspecific bands.





coactivator. It has been reported that the transcriptionalactivity of b-catenin is promoted by p300 (17) and thatp300 is recruited by TADs of HIF-as (18, 19). Therefore, weexamined p300 recruitment by b-catenin, and found that the

b-catenin–p300 binding was enhanced by full-length HIF-2a,but not by the d67 mutant (Fig. 4D). Summarizing, HIF-2aappears to act like a coactivator during b-catenin/TCF-driventranscription.

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Figure 3. HIF-2a enhances the transcriptional activity of b-catenin. A, effect of HIF-1/2a on b-catenin/TCF activity stimulated by LiCl. TOPflash,FOPflash, SV40-promoter, or VEGF-promoter luciferase plasmid was cotransfected into HEK293T cells with pCMV-b-gal and pHIF-1/2a. Next day, cells weretreated with 20 mmol/L LiCl for 16 hours. Luciferase activity was divided by b-gal activity to normalize transfection efficiency. Results are quoted as relativevalues versus the control value (n ¼ 1) and plotted as mean þ SD (n ¼ 12). *, P < 0.05 versus the LiCl-treated group; #, P < 0.05 versus the pcDNAcontrol. B, effect of HIF-1/2a on the activity of expressed b-catenin. HIF-1/2a or d67 mutant of HIF-2a were coexpressed with TOPflash, b-gal, andHA-b-catenin in HEK293T cells. Luciferase activities (n ¼ 4, mean þ SD) are quoted as relative values versus the control. *, P < 0.05 versus the HA-b-cateningroup. C, effects of HIF-2a fragments on the activity of expressed b-catenin. HEK293T cells were cotransfected with pTOPflash, pCMV-b-gal,pHA-b-catenin, and pFLAG-HIF-2a fragments. Luciferase activities (n¼ 12, meanþ SD) are quoted as relative values versus the control. *, P < 0.05 versus theHA-b-catenin group. D, HIF-2a enhances the expression of b-catenin target genes. HEK293T cells, which had been transfected with wild-type HIF-2aor d67 HIF-2a plasmid, were treated with 20mmol/L LiCl for 4 hours, and Axin2 and c-Myc were analyzed by immunoblotting. Protein levels were estimated byImageJ. Data (n ¼ 3, mean þ SD) are quoted as relative values versus the pcDNA control. *, P < 0.05 versus the LiCl-treated pcDNA group.

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Endogenous interaction between b-cateninand HIF-2a in 786-0 cellsThe findings described earlier suggest that HIF-2a in

association with b-catenin participates in tumor cell growth.We chose the VHL-deficient 786-0 cell line as an experi-mental model for examining the role of HIF-2a, because itexpresses HIF-2a exclusively. As previously reported (20),786-0 cells expressed HIF-2a even in normoxia. Interestingly,we also found that b-catenin and TCF4 were constitutivelypresent in nuclear fractions (Fig. 5A). Furthermore, immu-

nofluorescent analyses revealed that both HIF-2a andb-catenin colocalized in the nuclei (Fig. 5B). Given thatthe intensity of green fluorescence was weakened by siRNAof b-catenin, the fluorescence is regarded to be b-cateninspecific (Supplementary Fig. S5). In addition, immunopre-cipitation analyses showed that HIF-2a interacts withb-catenin endogenously (Fig. 5C). In 786-0 cells, TCF4 wasfound to coprecipitate with b-catenin in the absence of HIF-2a (Fig. 5D), which further supports the notion that HIF-2adoes not affect b-catenin/TCF4 binding.

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D

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Figure 4. HIF-2a TADs enhances the transcriptional activity of b-catenin by recruiting p300. A, HIF-2a does not affect nuclear translocation of b-catenin.After HEK293T cells were treated with 20 mmol/L LiCl for 4 hours, the nuclear proteins were extracted and analyzed by immunoblotting. B, HIF-2a does notaffect the formation of b-catenin/TCF4 complex. HA-b-catenin, Myc-TCF4, or HIF-2a was expressed in HEK293T cells. Immunoprecipitation wasperformed using anti-HA or anti-Myc, and precipitated proteins were identified by immunoblotting. C, HIF-2a enhances the b-catenin/TCF4-derivedtranscription through 2 TADs. HEK293T cells were cotransfected with pTOPflash, pCMV-b-gal, and 1 of pHIF-2a mutants. Luciferase activities (n ¼ 4,mean þ SD) are quoted as relative values versus the control. *, P < 0.05 versus the full-length HIF-2a group. D, p300 recruitment by HIF-2a. pHA-p300,pFLAG-b-catenin, and pHIF-2a full-length or pHIF-2a d67 were transfected, and immunoprecipitation was done with anti-HA.





HIF-2a is required for b-catenin activation andproliferation in 786-0 cells

b-catenin functions to promote cell growth in tumors eitherby inducing c-Myc or by repressing p21 (21). Because b-cate-nin and HIF-2a were found to associate in 786-0 cells, weexamined the role of HIF-2a in b-catenin signaling. In parti-cular, we used the gene silencing technique instead of ectopicexpression to investigate the role of endogenous HIF-2a. In786-0 cells, TOPflash activity was significantly attenuated byHIF-2a knockdown, whereas FOPflash and SV40-promoteractivities were constant regardless of HIF-2a level (Fig. 6A).Moreover, HIF-2a inhibition suppressed c-Myc and cyclin D1

but induced p21, which was reversed by HIF-2a overexpres-sion (Fig. 6B). During FACS analysis of the cell cycle, the G1/G0population was found to increase and the S population todecrease after HIF-2a silencing (Fig. 6C). Furthermore, cellproliferation (determined by BrdU incorporation) was foundto be significantly inhibited by HIF-2a silencing (Fig. 6D;Supplementary Fig. S6). Moreover, the population of prolifer-ating cells was significantly reduced by knocking down eitherb-catenin or TCF4 (Supplementary Fig. S7), suggesting thatb-catenin signaling is required for 786-0 cell growth. Takentogether, HIF-2a is likely to promote 786-0 cell cycle andproliferation by facilitating b-catenin signaling.

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Figure 5. b-catenin and HIF-2a naturally associate in 786-0 cells. A, b-catenin and HIF-2a are constitutively present in the nuclei of 786-0 cells.After 786-0 cells were cultured in the presence of LiCl for 4 hours, proteins were analyzed in the nuclear (Nu) and cytoplasmic (Cy) fractions by immunoblotting.B, HIF-2a and b-catenin colocalize in the nucleus; 786-0 cells were cultured onto cover glasses for 24 hours. Cells were fixed, permeated, and incubated withindicated primary antibodies for 16 hours, followed by incubation with ALEXA Fluor–conjugated secondary antibodies and DAPI. Fluorescence imageswere captured at �100 magnification. Red, HIF-2a; green, b-catenin; blue, DAPI. C, association of endogenous HIF-2a and b-catenin in 786-0 cells. In thenuclear extracts of 786-0 cells, b-catenin or HIF-2a was immunoprecipitated, and coprecipitated proteins were identified by immunoblotting. To verifythe specificity of anti–b-catenin antiserum, 786-0 cells were transfected with siRNA targeting b-catenin and subjected to immunoprecipitation analysis.D, HIF-2a is not required for b-catenin/TCF4 binding. After HIF-2a was knocked down in 786-0 cells using 40 nmol/L siRNA, b-catenin/TCF4 binding wasanalyzed by immunoprecipitation–immunoblotting.

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Discussion

In this study, we found that HIF-2a associates with b-cate-nin/TCF complex via an interaction between the N termini ofHIF-2a and b-catenin. Functionally, HIF-2a enhances thetranscriptional activity of b-catenin, which may be attributa-ble to HIF-2a TADs-driven transcription and to the increasedrecruitment of p300. Furthermore, HIF-2a was found to berequired for b-catenin activation and cell proliferation in 786-0ccRCC cells. These results suggest that the interplay betweenHIF-2a and b-catenin contributes to the unrestrained growthof ccRCCs, as characterized by the coactivation of HIF-2a andb-catenin. In particular, these actions of HIF-2a were found tooppose the inhibitory actions of HIF-1a on b-catenin and cellgrowth, and thus, a balance between HIF-as may importantlydetermine cell growth when hypoxia and Wnt stimulationcoexist.In addition to its role as a transcription factor, HIF-1a is

known to modulate diverse cell signaling by directly interact-ing with key signaling components. For instance, the inter-actions between HIF-1a and cell cycle–related proteins, such

as, p53 (22), c-Myc (7), b-catenin (11), and hARD1 (13), resultin cell cycle arrest and growth inhibition. On the contrary,HIF-2a has been reported to regulate the cell cycle andproliferation positively in some tumors (6, 23). As comparedwith the multiple partnerships of HIF-1a, however, c-Mycalone was found to be a cell cycle–related protein thatinteracts with HIF-2a. In a previous study (8), c-Myc/MAXbinding increased in parallel with HIF-2a expression, but thisbinding was dissociated by HIF-1a. This event was proposedto be the prime mechanism whereby HIF-as differentiallyregulate cell fate. We here found that b-catenin is another cellcycle–related protein differentially regulated by HIF-as. Theinactivation of b-catenin by HIF-1a has been previouslysuggested by Kaidi et al. (11). Mechanistically, HIF-1a inter-acts with b-catenin and this leads to TCF4 dissociation fromb-catenin, which is similar with the mode-of-action of HIF-1adisrupting the c-Myc/MAX complex. However, we found thatHIF-2a neither reverses HIF-1a binding to b-catenin noraffects b-catenin/TCF4 binding. These results suggest thatHIF-2a does not simply oppose HIF-1a in b-catenin regula-tion, rather acts as a coactivator for b-catenin/TCF-4.

A

CD

B

Figure 6. HIF-2a promotes b-catenin activation and cell proliferation in 786-0. A, the basal activity of b-catenin depends on HIF-2a in 786-0 cells. Afterbeing transfected with 40 nmol/L HIF-2a siRNA and 2 mg of pTOP/FOP-flash or pSV40-promoter reporter (SV40-Luc) for 48 hours, cells were lysed to analyzeluciferase activities, which were normalized by b-gal activities. TOPflash/FOPflash or SV40-Luc results (n ¼ 8, mean þ SD) are quoted as relative valuesversus the control value of TOPflash or SV40-Luc, respectively. *, P < 0.05 versus the control. B, HIF-2a silencing leads to c-Myc downregulation andp21 upregulation in 786-0 cells. After being treated with siRNAs for 48 hours, 786-0 cells were lysed for immunoblotting. C, HIF-2a silencing causescell cycle arrest. After being treated with siRNAs or/and HIF-2a plasmid for 48 hours, 786-0 cells were prepared to analyze cell cycle with FACS.*, P < 0.05 versus the control group; #, P < 0.05 versus the si-HIF-2a II group. D, HIF-2a silencing attenuates proliferation of 786-0 cells. After treatment withsiRNAs or/and HIF-2a plasmid for 48 hours, the cell proliferation was analyzed by BrdU incorporation and FACS. BrdU-positive and S-phase cellswere assessed as proliferating cells. *, P < 0.05 versus the control group; #, P < 0.05 versus the si-HIF-2a II group.





In VHL hereditary cancer syndrome, deletion or loss-of-function mutation of the VHL gene primarily causes highlyvascular tumors, such as, ccRCC, hemangioblastoma, andpheochromocytoma (24). In approximately 70% of sporadicccRCCs, VHL was found to be inactivated, and consequentlyHIF-a levels were elevated (25). However, HIF-1a and HIF-2aseem to contribute differently to tumor promotion in ccRCC.Some clinical studies have shown that HIF-2a has a tendencyto be expressed in the more advanced lesions of the VHLkidneys, and that HIF-1a elevation is associated with a betterprognosis in ccRCC (26, 27). In RCC xenograft studies, tumorsexpressingHIF-2a grew faster, and tumor growthwas retardedby HIF-2a knockdown. In contrast, HIF-1a expression inhib-ited xenograft growth (28). Therefore, HIF-2a appears to bemore critical for ccRCC progression. Considering that b-cate-nin is activated by loss of VHL, HIF-2a stimulation of b-cateninis regarded to have a substantial impact on ccRCC growth.

In ccRCC cells, b-catenin levels are inversely correlated withfunctional VHL levels. Studies that have examined VHLexpression and silencing have shown that VHL repressesb-catenin/TCF-driven transcription by destabilizing b-cate-nin. Mechanistically, it has been shown that VHL proteinbinds and stabilizes Jade-1, and that this leads to the ubiqui-tination and subsequent degradation of b-catenin (29), whichsuggests that b-catenin activation due to VHL loss may beassociated with the aggressive behavior of ccRCC. In thisstudy, we found that b-catenin is constitutively activated in786-0 ccRCC cells, and that this might be caused by VHL lossand the expression of HIF-2a, but not of HIF-1a, by these cells.Accordingly, we regarded 786-0 as a suitable cell line toexamine the interaction between HIF-2a and b-catenin,and consequently identified a novel molecular link betweenthe 2 RCC-relevant pathways.

In many cases, cancer cells express both HIF-1a and HIF-2ain response to hypoxia. A recent report showed that HIF-asare differentially regulated by hypoxia (30). According to thisreport, HIF-2a in neuroblastoma specimens was frequentlydetected in oxygenated regions around blood vessels, whereas

HIF-1a was not. Furthermore, in culture, HIF-2a was sub-stantially stabilized by 5% oxygen, whereas HIF-1a was sta-bilized only at an oxygen level of 1%. These findings suggestthat HIF-2a contributes to hypoxic response under mildhypoxia and that HIF-1a does so under severe hypoxia. Ourresults and this information suggest that cancers keep grow-ing during tolerable hypoxia by utilizing HIF-2a but stopgrowing during intolerable hypoxia by utilizing HIF-1a. How-ever, further experiments are required to confirm this scenarioto improve our understanding of tumor fate under hypoxicconditions.

This study shows that HIF-2a binds b-catenin andenhances the transcriptional activity of b-catenin/TCF byrecruiting p300. Moreover, it shows that HIF-2a in associationwith b-catenin contributes to cell cycle progression andproliferation in 786-0 cells. In addition to its role in c-Mycsignaling, the activation of b-catenin signaling by HIF-2amaycontribute to tumor growth in VHL�/� RCC or in hypoxictumors. In addition to cell growth, HIF-2a upregulates manygenes critical for angiogenesis, metastasis, and dedifferentia-tion, which are all related to a poor outcome, which suggeststhat HIF-2amight be a useful biomarker for evaluating cancerstates and a therapeutic target, especially in ccRCC and othertumors that highly express HIF-2a.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the Korea Healthcare technology R&D Project,Ministry for Health, Welfare & Family Affairs, Republic of Korea (A091081 andA090685), and by a grant from the Seoul National University Hospital ResearchFund (2010).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 02/09/2010; revised 09/22/2010; accepted 10/06/2010; publishedOnline 12/15/2010.

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2010;70:10101-10111. Cancer Res Hyunsung Choi, Yang-Sook Chun, Tae-You Kim, et al.

-CateninβInteracting with -Catenin/TCF-Driven Transcription byβ Enhances αHIF-2

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