JAG2 Induction in Hypoxic Tumor Cells Alters Notch ... · resulted in elevated Notch activity in...

DNA Damage and Cellular Stress Responses

JAG2 Induction in Hypoxic Tumor Cells Alters Notch Signalingand Enhances Endothelial Cell Tube Formation

Alexander Pietras1,2, Kristoffer von Stedingk1, David Lindgren1, Sven Pa�hlman1,2, and Ha

�kan Axelson1

AbstractSeveral studies have revealed links between hypoxia and activation of Notch in solid tumors. While most reports

have focused on intracellular domain of the Notch1 receptor (icN1) stabilization by direct interaction with HIFproteins, little attention has been given to Notch ligand regulation during hypoxia. Here we show that the Notchligand JAG2 is transcriptionally activated by hypoxia in a HIF-1a dependent manner. Hypoxic JAG2 inductionresulted in elevated Notch activity in tumor cells, as was measured by increased icN1 levels and induction of theNotch target gene HEY1. In primary tumor material, JAG2 expression correlated with vascular development andangiogenesis gene signatures. In line with this, coculture experiments of endothelial cells with hypoxic breastcancer cells displayed a reduction in number of capillary-like tubes formed upon JAG2 siRNA treatment of thebreast cancer cells. Together these results suggest that a hypoxic induction of JAG2 in tumor cells mediates ahypoxia-regulated cross-talk between tumor and endothelial cells. Mol Cancer Res; 9(5); 626–36. �2011 AACR.

Introduction

During growth and expansion of solid tumors, thenutrient and oxygen requirements of the expanding tumorgenerally surpass that being supplied by the supportingvasculature, leading to environmental oxygen shortage (1).This results in a cellular hypoxic response, primarily orche-strated by the hypoxia-inducible factors (HIF; ref. 2). HIFheterodimers consist of a-subunits (HIF-1a and HIF-2a)and the constitutively stable HIF-1b subunit [ARNT (arylhydrocarbon receptor nuclear translocator)]. HIF-a andHIF-b subunits heterodimerize within the nucleus wherethey act as transcription factors, binding to hypoxicresponse elements (HRE) in HIF target genes such asvascular endothelial growth factor (VEGF; ref. 3, 4). Viathe induction of VEGF, among others, the hypoxic responseinitiates tumor neoangiogenesis. This process is a result ofendothelial cell proliferation, migration, and differentiationalong the VEGF gradient produced by the tumor (1). Inaddition to VEGF regulation of vascular development,branching and expansion of the vasculature are under thecontrol of cell–cell activation of Notch signaling (5). Notch

signaling is activated by membrane-bound ligand presenta-tion to a Notch receptor on adjacent cells. Notch ligandsinclude the delta-like and jagged ligands (DLL1, DLL3,DLL4, JAG1, and JAG2, respectively). Interaction of theseligands with one of the Notch receptors (NOTCH1-4)results in g-secretase–mediated cleavage and release of theintracellular domain of the Notch receptor (icN). icNfunctions as a nuclear transcription cofactor to activateNotch target genes, such as genes of the Hairy/Enhancerof Split (HES) and Hes-related (HEY) families of proteins(6) through interaction with the CBF1/Su(H)/Lag-1 (CSL)[recombination signal binding protein for immunoglobulinkappa J region (RBPJ)] transcription factor. During vasculardevelopment, the Notch ligand DLL4 is of particularimportance because DLL4-mediated activation of Notchsignaling in vasculature results in a process known as lateralinhibition; a process vital for modulating branch formationduring angiogenesis (7).In hypoxic cells cross-talks between HIF and Notch

pathways have been reported, primarily resulting in ele-vated Notch signaling. Several mechanisms have beenproposed including direct icN1 stabilization by interac-tion with HIF proteins (8–18). As of yet, little attentionhas been given to Notch ligand regulation and the effectsthis may have on Notch activation under hypoxia.Furthermore, whether hypoxia-induced Notch signalingmay influence tumor angiogenesis remains to be investi-gated. Here we show that hypoxia resulted in elevatedexpression levels of the Notch ligand JAG2 via transcrip-tional activation by HIF-1a. This elevation was respon-sible for a substantial part of the increased Notch activityobserved in hypoxic tumor cells. JAG2 expression inprimary tumor material was correlated with gene signa-tures of vascular development and angiogenesis. In addi-tion, coculture of vascular endothelial cells with hypoxic

Authors' Affiliations: 1Center for Molecular Pathology and 2CREATEHealth, Department of Laboratory Medicine, Lund University, Ska

�ne Uni-

versity Hospital, Malm€o, Sweden

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

A. Pietras and K. von Stedingk contributed equally to this work.

Corresponding Author: Ha�kan Axelson, Center for Molecular Pathology,

Department of Laboratory Medicine, Ska�ne University Hospital, Entrance

78, SE-205 02 Malm€o, Sweden. Phone: 46-40337621; Fax: 46-40337063.E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0508

�2011 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 9(5) May 2011626

Research. on February 17, 2020. © 2011 American Association for Cancermcr.aacrjournals.org Downloaded from

Published OnlineFirst March 14, 2011; DOI: 10.1158/1541-7786.MCR-10-0508

http://mcr.aacrjournals.org/

tumor cells revealed that JAG2 expression on tumor cellspromoted endothelial cell tube formation. Together theseresults suggest that a hypoxia-regulated cross-talk betweentumor and endothelial cells via Notch activation isinvolved during tumor angiogenesis.

Materials and Methods

Cell cultureTumor cells. MCF7 and T47D cells [ATCC (American

Type Culture Collection)] were cultured in RPMI-1640growth medium, with 10% FBS and 1% penicillin-strepto-mycin (PEST). T47D cell medium also contained 1%glucose, 1% sodium pyruvate, and 5 mL/mL insulin. Undernormoxic conditions cells were cultured in a humidifiedincubator maintained at 37�C with 5% CO2 and 95% air(approximately 21% O2). Under hypoxic conditions thecells were cultured in a hypoxystation H35 cell culture in-cubator, maintained at 37�C with 1% O2, 5% CO2, and94% N2.Endothelial cells. MS1 murine endothelial cells were

cultured at the same normoxic conditions as describedabove in EBM-2 medium (Lonza) supplemented withEGM-2 MV SingleQuots (Lonza; hydrocortisone, hEGF,FBS, VEGF, hFGFB, R3-IGF-1, ascorbic acid, and genta-micin/amphotericin-B). The cell culture plates were coatedwith 1% gelatin.

siRNA transfections for quantitative PCR and proteinanalysisCells were seeded out into 6 well plates (2 wells per si

treatment) at a density of 120,000 cells per well. The cellswere left to attach overnight. The cell medium (asdescribed above) was removed and replaced with 2 mLOptimem (Invitrogen) without any added FBS or anti-biotics. Lipofectamin 2000 (Invitrogen) was used as thetransfection agent. Predesigned Silencer Select siRNAs(Ambion) were used to silence desired genes. Tranfectionswere carried out with a Lipofectamin 2000 concentrationof 1.5 mL/mL, and siRNA concentrations ranging from2.5 to 10 nmol/L. Cells were tranfected under normoxicconditions (described above) for 6 hours. The transfectionmedium was then replaced with respective cell mediums(described above) containing 10% FBS and no antibiotics.Cells were maintained in normoxic culture overnight.Cells were then moved to respective oxygen conditions(21% or 1% O2) and cultured for 24 hours beforeharvesting.

siRNA transfections for coculture experimentsCells were seeded out into as above at a density of

200,000 cells per well. Cells were left to attach overnight.The transfection procedure was carried out as above. Fol-lowing the 6-hour transfection, the transfection mediumwas replaced with respective cell mediums (describedabove), and the cells were moved to respective oxygenconditions (21% or 1% O2). The cells were culturedovernight before seeding in the coculture experiments.

Chromatin immunoprecipitationT47D cells were cultured under hypoxia for 24 hours.

Cells were then harvested and immunoprecipitation wascarried out using the Diagenode Transcription Factorchromatin immunoprecipitation (ChIP) Kit according toprotocol. In brief, the cells were fixed and collected using thescraping method. This was followed by cell lysis in 1�proteosome inhibitor containing lysis buffer. Chromatinshearing was then carried out using a Diagenode Bioruptorsonicator set on high for 7.5 cycles of 30 seconds at 4�C.Immunoprecipitation was carried out using either anti-human IgG antibody (rabbit polyclonal, Abcam) or anti-human HIF-1a antibody (rabbit polyclonal—ChIP grade,Abcam). Precipitated protein–DNA complexes were thende-cross-linked by overnight incubation at 65�C in athermoshaker. DNA was purified using phenol extractionand used for quantitative real-time PCR (q RT-PCR) usingprimers specific for a potential HIF binding sites within theJAG2 promoter (Supplementary Table S1B). Primers tar-geting the known HRE within the VEGF promoter wereused as the positive control, and primers targeting a regionupstream of the JAG2 promoter were used as a negativecontrol (Supplementary Table S1B). Relative precipitatedDNA values were calculated using the Delta-Delta-CTMethod.

Quantitative real-time PCRCells were lysed and homogenized using the QIAsh-

redder Kit (Qiagen), then loaded onto RNeasy spincolumns (Qiagen) for total RNA extraction accordingto the manufacturer's recommendation. After DNAsetreatment and extensive washing using Microcon spincolumns, cDNA was synthesized using random primersand Multiscribe Reverse Transcriptase enzyme (AppliedBiosystems). Generated cDNA was used as template withSYBR Green PCR Master Mix (Applied Biosystems) andexpression levels were quantified using the comparative Ctmethod. Expression levels were normalized using theexpression of 3 housekeeping genes (UBC, YWHAZ,and SDHA; ref. 19). Primer sequences are listed inSupplementary Table S1A.

Western blotFor protein extraction, cells were pelleted by centrifuga-

tion, then lysed in 10 mmol/L Tris-HCL (pH 7.2), 160mmol/LNaCl, 1%TritonX-100, 1% sodiumdeoxycholate,0.1% SDS, 1 mmol/L EGTA, and 1 mmol/L EDTA [RIPA(radioimmunoprecipitation assay buffer)] supplementedwithComplete Protease Inhibitor cocktail (RocheMolecularBiochemicals). The method of Bradford was used to deter-mine protein concentrations. SDS-PAGE separation ofproteins in MOPS buffer (Invitrogen) and subsequentblotting onto polyvinylidene difluoride (PVDF) membranes(Millipore) was done using the NuPAGE gels and system(Invitrogen) according to the manufacturer's instructions.Membrane blocking and antibody dilutions were made in5%milk diluted in 0.010 mol/L phosphate buffer (pH 7.4),2.7 mmol/L KCl, 0.140 mol/L NaCl, 0.05% Tween 20

Hypoxia-Induced JAG2 in Breast Cancer

www.aacrjournals.org Mol Cancer Res; 9(5) May 2011 627




(PBS-Tween). The following antibodies and antisera wereused: actin (MP Biomedicals; diluted 1:2,000), IntracellularNotch-1 (Cell Signaling; diluted 1:500), JAG2 (Abcam;diluted 1:400), and sheep anti-mouse and anti-rabbitsecondary antibodies (GE Healthcare; diluted 1:5,000).

Immunohistochemistry and immunofluorescenceFor immunohistochemistry, human ductal carcinoma in

situ (DCIS) specimens were routinely embedded in paraffinupon fixation. The Envision system and DAKO Techmate500 were used for detection of protein after incubation ofsections with the following primary antibodies: HIF-1a(Upstate; diluted 1:100) and JAG2 (Abcam; diluted 1:250).For immunofluorescence, cells were grown on cover slips

and then fixed in 4% paraformaldehyde. After blocking in5% normal goat serum and 0.3% Triton X-100 in PBS for30 minutes, cover slips were incubated for 1 hour withJAG2 antisera (Abcam; diluted 1:400) diluted in PBS, thenwashed and further incubated for 1 hour with secondaryantibody conjugated to Alexa fluor 488 (Molecular Probes).Cover slips were mounted in PVA-DABCO.

Endothelial cell tube formationTen thousand MS1 (transformed mouse pancreatic islet

endothelial) cells (20; a kind gift from Dr. Kristian Pietras,Karolinska Institutet, Stockholm, Sweden) were mixedwith 104 T47D breast cancer cells (treated as indicated),then seeded in 96 well plates on top of a layer of growthfactor-reduced Matrigel (BD) in EBM-2 medium (Lonza)supplemented with EGM-2 MV SingleQuots (Lonza;hydrocortisone, hEGF, FBS, VEGF, hFGFB, R3-IGF-1,ascorbic acid, and gentamicin/amphotericin-B). Matrigelwas thawed slowly at 4�C overnight, then allowed topolymerize at 37�C for 30 minutes before seeding. Tubeformation and length was measured using 3 photographsfrom each group taken at 8 hour postseeding. For number oftubes formed, each connected tube in each photograph wascounted and average values within groups were compared.For measurements of tube length, images were first pro-cessed using the ImageJ software and the Tubeness plugin(21) to visualize tubes. Each tube was then identified andtotal tube length quantified using the Fiji software (http://pacific.mpi-cbg.de/wiki/index.php/Main_Page) and theSimple neurite tracer plugin (http://homepages.inf.ed.ac.uk/s9808248/imagej/tracer/). In both cases, values werenormalized to the average value in cells cultured withuntransfected T47D cells and statistical significance calcu-lated using a one-way ANOVA followed by Tukey HSD(honestly significant difference) test comparing the controltransfected group with JAG2 siRNA groups. For condi-tioned media experiments, siRNA-treated T47D cells werecultured in EBM2 medium overnight under hypoxia, afterwhich MS1 cells were seeded as above with conditionedmedia replacing the T47D cells.

Nuclear extractsNuclei were extracted after harvesting cells in 40 mmol/L

Tris-HCl pH 7.5, 1 mmol/L EDTA, 150 mmol/L NaCl

(TEN), then washed in PBS and 25 mmol/L Tris pH 7.5,50 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L EDTA, 5mmol/L Dithiotreitol (DTT; Buffer A). Pellets were resus-pended in 25 mmol/L Tris pH 7.5, 0.42 mol/L NaCl, 1.5mmol/L MgCl2, 0.5 mmol/L EDTA, 1 mmol/L DTT,25% Sucrose (Buffer NE) and nuclei extracted for 300. Aftercentrifugation to remove cellular debris, supernatants wereused as the nuclear fraction.

Gene expression analysisJAG2 expression data from Chi and colleagues (22) were

analyzed using Oncomine (23). For the JAG2 correlation

Oxygen %102 kD-

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Figure 1. Notch signaling is elevated in hypoxia. MCF7 and T47D breastcancer cells were cultured in normoxia (21% oxygen) and hypoxia (1%oxygen). A, Western blot analysis of activated NOTCH1 (icN1) on nuclearextracts. B, QPCR analysis of mRNA expression levels of HEY1.Expression levels are displayed as relative expression levels to thoseobserved in normoxia. Data represent 1 of at least 3 independentexperiments run in triplicate. Error bars represent SD. C, relative mRNAlevels of the Notch receptors (NOTCH1-4). D, relative mRNA levels of theNotch ligands (DLL1, DLL4, JAG1, JAG2). DLL3 transcript levels are notdisplayed due to Ct levels being below the detection cutoff point of 35.Average normoxic Ct values obtained from QPCR analysis are displayed.Error bars represent SD. Data represent mean values from 3 independentexperiments run in triplicate. *, indicates P < 0.05; **, indicates P < 0.01, in a2-tailed unpaired Student's t test.

Pietras et al.

Mol Cancer Res; 9(5) May 2011 Molecular Cancer Research628




analyses in primary tumor material, 2 gene expression datasets, the first comprising 200 breast tumors (24) and a secondcomprising 59 clear cell renal carcinoma samples (25) weredownloaded from the NCBI (National Center for Biotech-nology Information) GEO website (http://www.ncbi.nlm.nih.gov/geo/; accession numbers GSE11121 andGSE14994, respectively). RawCEL files were RMAnormal-ized in R (http://www.r-project.org) using the Affy package.For both of these data sets, the Pearson correlation wascalculated between JAG2 expression and all other reporters

present on the array. Genes were then ranked according totheir respective JAG2 correlations and gene set enrichmentanalysis (GSEA; ref. 26) was applied on these ranked listsusing the c2.v2.5 curated gene sets collection as supplied bythe Molecular Signatures Database (MsigDB; broad.mit.edu/gsea/msigdb). Gene Ontology (GO) analyses were car-ried out on the 100 reporters showing highest correlation toJAG2 using the Functional AnnotationTool inDAVID (27,28) and on the complete ranked JAG2 correlation lists usingthe c5.v2.5 GO gene sets collection in GSEA.

21% oxygen

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Figure 2. JAG2 protein and mRNA levels are elevated by hypoxia in vivo and in vitro in different tissues/cells. A, immunofluorescence staining of JAG2 proteinin T47D cells cultured at normoxia or hypoxia. Inset shows immunohistochemical staining of JAG2 in hypoxic T47D cells. B, immunohistochemical analysisof a breast ductal carcinoma in situ. Antibodies recognizing HIF-1a (left) and JAG2 (right) were used. *, indicates areas magnified in insets. C, JAG2mRNA expression from public microarray data sets obtained from Oncomine. Expression analysis was carried out on mammary epithelial cells (left) and renalproximal tubule epithelial cells (right) that had been cultured at normoxia, hypoxia or anoxia. D, JAG2 mRNA expression from public microarray data sets oncultured human macrophages (44) and P493-6 Burkitt's lymphoma cells (45) and QPCR data on cultured human neuroblastoma SK-N-BE(2)C cells atnormoxia and hypoxia as indicated. Error bars represent SD. E, gene set enrichment analyses (GSEA) of hypoxia-upregulated gene set carried out onJAG2 correlated ranked gene lists produced from microarray analyses of 200 breast tumors (left; FDR q ¼ 0.009, NES: 1.99) or 59 RCCs (right; FDRq < 0.000, NES: 2.32).






JAG2 expression in hypoxic MCF7 cells treated withsiRNA targeting HIF1A and/or HIF2A was obtained fromthe previously published data set by Elvidge and colleagues(29). This data set was downloaded from the NCBI GEOwebsite with accession number GSE3188.

Results

Notch signaling is enhanced by hypoxiaMCF7 and T47D breast cancer cell lines were cultured at

21% or 1% oxygen (normoxia and hypoxia respectively) for

1.5

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HIF2A

Figure 3. Hypoxic JAG2 induction is HIF dependent. T47D cells were cultured at 21% or 1% oxygen and were treated with control siRNA or siRNA targetingHIF1A orHIF2A (A, B, C). mRNA levels were then examined using QPCR and presented as relative expression comparedwith normoxic levels. A, relativemRNAlevels of HIF1A. Data represent mean values from 3 independent experiments run in triplicate. Error bars represent SD. B, relative mRNA levels of HIF2A.Data represent mean values from 3 independent experiments run in triplicate. Error bars represent SD. C, relative mRNA levels of JAG2. Data represent meanvalues from 3 independent experiments run in triplicate. Error bars represent SD. *, indicates P < 0.05 in a 2-tailed unpaired Student's t test. D, JAG2 levelsin hypoxic MCF7 cells treated with siRNA targeting HIF1A and/or HIF2A as indicated, from data published in (29). Error bars represent SD. E, chromatinimmunoprecipitation of potential HRE located downstream of the JAG2 starting ATG carried out on T47D cells cultured under hypoxia for 24 hours.Pull-downswere carried out using anti-human IgG (negative control) and anti-humanHIF-1a. VEGF-promoter was used as a positive control and a nonenrichedregion in the JAG2 promoter as a negative control for the HIF-1a pull-down. Graph displays fold enrichment of precipitated DNA relative to backgroundIgG-pulled DNA. Data represent mean values from 2 independent experiments run in triplicate. Error bars represent SD of the fold enrichment.F, a schematic representation of the potential HREwithin JAG2. The sequence of the potential HIF-binding site is displayed and is present at theþ1294 position.

Pietras et al.





24 hours. In both cell lines, exposure to hypoxia resulted inincreased nuclear icN1 protein levels as indicated by westernblot analysis using an antibody specific for the activatedicN1 receptor (Fig. 1A). In conjunction with the increasedicN1 levels, expression of the known Notch target geneHEY1 (30) was also substantially elevated, as revealed byQPCR (Fig. 1B). These results are in agreement with thenotion that hypoxia enhances Notch signaling.

JAG2 is transcriptionally activated under hypoxiaTo determine potential factors responsible for the

increased Notch activation observed under hypoxia, theexpression of all Notch receptors and ligands in breastcancer cells were examined using QPCR both at normoxiaand hypoxia (Fig. 1C and D). With regards to the Notchreceptors, NOTCH1, NOTCH2, and NOTCH4 did notappear to be transcriptionally regulated by hypoxia, butNOTCH3 displayed increased transcript levels (Fig. 1C). Ithas previously been shown that NOTCH3 can be involvedin regulating the activity of icN1 and downstreamNOTCHtargets (31, 32), and can therefore not be excluded fromhaving an influence on icN1 elevation in hypoxia or effecton overall NOTCH signaling.The increase in cleaved icN1 levels could also potentially

be attributed to changes in Notch ligand expression. Expres-sion analyses revealed that although all Notch ligands, withthe exception of DLL3, which fell below detection limits,appeared upregulated under hypoxia, only JAG2 levels weresignificantly increased (Fig. 1D). Notably, the detectionlevel of the Notch ligand JAG2 was a minimum of 5 PCRcycles lower than that of all other ligands, suggesting highestexpression levels of JAG2 amongst the Notch ligands.

JAG2 is induced by hypoxia in vivo and in vitro indifferent tissues/cellsTo confirm that the increase in JAG2 observed at the

transcriptional level corresponded to an increase at theprotein level, JAG2 protein expression was examined byimmunofluorescence in T47D cells cultured at normoxia orhypoxia. The hypoxic T47D cells displayed an evidentinduction of JAG2 protein when compared with thosegrown at normoxia (Fig. 2A). The detected JAG2 proteinappeared to be primarily localized to the cellular membrane(Fig. 2A, inset) as was expected for a functional Notchligand. In addition, immunohistochemistry was carried outon sections of breast DCIS specimens, which in the comedoform are characterized by necrotic cores with surroundinghypoxic cells (33). JAG2 protein expression was elevated inthe hypoxic regions of the tumor surrounding the necroticcores (Fig. 2B), which was confirmed by detection of HIF-1a protein in the same perinecrotic regions.To determine if the hypoxic induction of JAG2 was

specific to breast cancer or if it could be detected in othercellular systems, expression data from hypoxia-treated cellswere examined using the online database Oncomine (23).Here it was found that for both mammary epithelial andrenal proximal tubule epithelial cells (22), exposure tohypoxia resulted in increased levels of JAG2 (Fig. 2C).Furthermore, microarray data from macrophage andBurkitt's lymphoma cells as well as QPCR data on neuro-blastoma cells cultured at normoxia and hypoxia showed aconsistent upregulation of JAG2 mRNA levels underhypoxic conditions (Fig. 2D).Moreover, we analysed a data set from a microarray

carried out on 200 breast cancers (24) as well as a data

Figure 4. JAG2 modulates Notchsignaling under hypoxia. T47Dcells were cultured at 21% or 1%oxygen and were treated withcontrol siRNA or siRNA targetingJAG2. A, relative mRNA levels ofJAG2were examined using QPCRand presented as relativeexpression compared withnormoxic levels. Data representmean values from 3 independentexperiments run in triplicate. Errorbars represent SD. B,Western blotanalysis of JAG2 in control andJAG2 siRNA-treated hypoxicT47D cells. C, Western blotanalysis of icN1 on nuclearextracts from untreated, control,and JAG2 siRNA-treated hypoxicT47D cells. D, QPCR-derivedrelative mRNA levels of HEY1,displayed as relative quantitiescompared with control-treatednormoxic T47D cells. Datarepresent mean values from 3independent experiments run intriplicate. Error bars represent SD.*, indicates P < 0.05, in a 2-tailedunpaired Student's t test.

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set consisting of 59 renal cell carcinomas (RCC; ref. 25). Foreach data set, all genes present on the arrays were rankedaccording to their correlation to JAG2 expression. GSEAwas then applied on the resulting JAG2 correlation rankedgene lists. Consistent in both the breast cancer and RCCarrays, the ranked lists displayed significant enrichment ofhypoxia-upregulated gene signatures (Fig. 2E; Supplemen-tary Table S2; FDR q ¼ 0.009, NES: 1.99; breast cancerand FDR q < 0.000, NES: 2.32; RCC). Together these datashow that hypoxia consistently results in increased JAG2mRNA and protein levels in a variety of cell types, both invivo and in vitro, and that JAG2 expression in both breastcancer and RCC correlated with transcriptional responsesobserved under hypoxia, suggesting that JAG2 induction isa general phenomenon in hypoxic cells.

JAG2 upregulation by hypoxia is HIF dependentTo determine if the hypoxic induction of JAG2 was HIF

dependent, T47D cells were cultured under hypoxia and

treated with siRNAs targeting either HIF1A or HIF2A(Fig. 3A and B). JAG2 levels were then monitored usingQPCR showing that knockdown of HIF1A, but notHIF2A, diminished JAG2 expression under hypoxia tolevels near that detected at normoxia (Fig. 3C). In addi-tion, a publicly available microarray data set on MCF7cells treated with siRNAs targeting HIF1A and/or HIF2Aunder hypoxia (29) also displayed a marked reduction inJAG2 levels upon HIF1A but not HIF2A silencing(Fig. 3D). To further confirm HIF-1a involvement inJAG2 induction, chromatin immunoprecipitation experi-ments were carried out on T47D cells cultured underhypoxia for 24 hours. As a positive control, HIF-1a pull-down on a previously defined VEGF-HRE (34) displayeda 5-fold enrichment compared with the backgroundIgG pull-down (Fig. 3E), and as a negative control, aregion upstream of the JAG2 promoter displayed noapparent enrichment (Fig. 3E). After screening a varietyof potential HREs located within the promoter region of

Table 1. JAG2 expression significantly correlates with angiogenic processes

Cancer, n Correlated processes P Benjamini(corrected P)

Breast, 200 Enrichment score: 4.85Blood vessel morphogenesis 6.0E-8 3.1E-4Blood vessel development 1.7E-7 4.6E-4Vascular development 2.0E-7 3.5E-4Angiogenesis 2.7E-7 3.5E-4Anatomical structure development 1.2E-6 1.3E-3Anatomical structure formation 1.5E-6 1.3E-3Anatomical structure morphogenesis 2.1E-6 1.6E-3Organ development 2.9E-6 1.9E-3Developmental process 4.0E-6 2.3E-3Organ porphogenesis 1.7E-5 9.1E-3Multicellular organismal development 6.4E-5 2.6E-2System development 7.7E-5 2.8E-2Multicellular organismal process 1.3E-3 2.2E-1Biological regulation 4.3E-3 4.7E-1Regulation of biological process 6.7E-3 6.0E-1Regulation of cellular process 2.9E-2 9.5E-1

RCC, 59 Enrichment score: 6.12Blood vessel development 1.1E-14 5.8E-11Vascular development 1.4E-14 3.6E-11Blood vessel morphogenesis 1.5E-12 2.7E-9Angiogenesis 4.0E-9 5.3E-6Organ morphogenesis 5.5E-9 5.8E-6Anatomical structure development 6.2E-9 5.4E-6Organ development 6.4E-9 4.8E-6Anatomical structure formation 2.9E-8 1.9E-5Anatomical structure morphogenesis 2.9E-8 1.7E-5Multicellular organismal development 4.1E-8 2.2E-5System development 3.3E-7 1.4E-4Developmental process 7.6E-7 3.1E-4Multicellular organismal process 1.4E-6 5.3E-4Angiogenesis 2.5E-5 4.5E-3

Pietras et al.





JAG2, HIF-1a pull-down yielded a 12-fold enrichmentover the negative control IgG. HIF-1a was found to bindstrongest within a sequence ranging from 1,229 to 1,328base-pairs downstream of the starting ATG of the JAG2promoter, with a potential HRE at the þ1294 position(Fig. 3F). This identified a potential HIF-1a responsiveHRE within the JAG2 gene, as well as identified JAG2 as adirect HIF target gene.

JAG2 contributes to the enhanced Notch activityobserved under hypoxiaTaking into consideration the elevated Notch signaling

observed during hypoxia, the role of JAG2 in hypoxicNotch activation was investigated. T47D cells treated withsiRNA targeting JAG2 were cultured in normoxia orhypoxia. The JAG2 knockdown was confirmed at bothmRNA and protein levels under hypoxia (Figs. 4A and1B). To examine the effects of diminished JAG2 levels onNotch signaling, icN1 protein levels were again examinedby Western blot. The knockdown of JAG2 resulted in asubstantial decrease in nuclear icN1 protein levels presentunder hypoxia (Fig. 4C). In addition, the Notch targetgene HEY1 also displayed marked reduction in its hypoxicupregulation upon JAG2 depletion (Fig. 4D). Consideringthe reduction in both cleaved NOTCH1 levels andhypoxic HEY1 induction, JAG2 elevation strongly con-tributed to the increase in Notch activity observed underhypoxia.

JAG2 expression signature in primary tumor materialsignificantly correlates with angiogenic processesTo further investigate which functional processes are

associated with JAG2 expression in primary cancer materi-als, a GO analysis was carried out on the 100 array reportersshowing highest correlation to JAG2 expression. Interest-ingly, in both breast cancer and RCC arrays, several angio-genic ontology terms were significantly enriched among theJAG2 correlated genes (Table 1), including processes such asblood vessel morphogenesis, blood vessel development,vascular development, and angiogenesis. GO analysis wasalso carried out on the full JAG2 correlation ranked listsusing GSEA, again displaying enrichments for vasculardevelopment (Fig. 5A and B; Supplementary Table S2)and angiogenesis (Fig. 5A and B; Supplementary Table S2)in both data sets (FDR q¼ 0.009, NES: 1.97; breast cancervascular development; FDR q ¼ 0.023, NES: 1.86; breastcancer angiogenesis; FDR q ¼ 0.04, NES: 1.79; RCCvascular development; FDR q ¼ 0.05, NES: 1.75; RCCangiogenesis).

Hypoxic JAG2 expression in epithelial tumor cellscontributes to endothelial cell tube formationBecause Notch signaling has a pivotal role in endothelial

cell angiogenesis and recent work has displayed interactionsbetween tumor epithelial cells and their surrounding vas-cular endothelial cells (35), we next investigated whetherepithelial JAG2 upregulation under hypoxia might have a

Figure 5. JAG2 correlated geneexpression displays enrichmentfor angiogenic processes. A,GSEAs carried out on a JAG2correlated ranked gene listproduced from a microarrayexamining gene expression in 200breast cancers. Significantenrichments were observed forboth vascular development (left;FDR q ¼ 0.009, NES: 1.97) andangiogenesis (right; FDR q ¼0.023, NES: 1.86) gene signatures.B, GSEAs carried out on a JAG2correlated ranked gene listproduced from a microarrayexamining gene expression in 70RCC. Enrichments were observedfor both vascular development(left; FDR q¼ 0.04, NES: 1.79) andangiogenesis (right; FDR q¼ 0.05,NES: 1.75) gene signatures.

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direct role in regulating tumor angiogenesis. To investigatethis, an in vitro coculture system was employed. T47D cellstreated with control or JAG2 siRNA were cultured underhypoxia for 18 hours before being seeded onto Matrigel incombination with the murine-derived endothelial cells MS1(20). The tube formation capacity of the endothelial cellswas then examined (Fig. 6A). Interestingly, depletion ofJAG2 from the epithelial T47D cell population resulted in asignificant reduction in MS1 cell tube formation measuredas number of tubes formed as well as total tube length(Fig. 6B and C). This reduction appeared cell–cell contactdependent, as MS1 cells grown with conditioned mediumfrom siRNA-treated hypoxic T47D cells displayed no sig-nificant change in tube formation between groups(Fig. 6D).

Discussion

It has been previously reported that Notch signaling iselevated during hypoxia (8, 10, 11, 14, 18). Here we showthat a substantial part of this elevation can be attributed toincreased expression of theNotch ligand JAG2. The hypoxicinduction of JAG2 was HIF dependent, and we show HIFbinding to a potential HRE downstream of the JAG2 startcodon. In human tumors, JAG2 protein levels were elevatedin hypoxic regions of the tumor, and GSEA analysis revealed

that JAG2 expression is correlated with hypoxic gene sig-natures in primary breast cancer and RCC material.Until now, reports on Notch activity in hypoxia have

largely focused on intracellular interactions between icNand HIFs, resulting in enhanced transcription of Notchdownstream targets (8, 10). Even though our results showedthat JAG2 was responsible for a large fraction of the Notchactivity under hypoxia, silencing of JAG2 did not result incomplete inhibition of the hypoxic potentiation of Notchsignaling. In light of the previous reports, the remainingelevation in Notch activity could be due to direct HIF-icN1interactions or elevation of other Notch receptors (36).The development of a hypoxic environment inevitably

results in initiation of angiogenesis and recruitment of newblood vessels. It has been shown that hypoxic cells secreteVEGF among other growth factors, creating a VEGFgradient between the hypoxic source and the surroundingvasculature (1). The vasculature is then activated by thesecreted factors and extends in the direction of the VEGFgradient (37). The mechanism behind the extension/for-mation of the new vasculature has been a recent subject ofintense interest and has led to development of VEGFinhibitors for antiangiogenic tumor therapies. Recent stu-dies have also unveiled the pivotal importance of Notchsignaling in the process of angiogenesis, with specific focuson 2 Notch ligands DLL4 and JAG1 (5, 38–42). It is

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Figure 6. Tumor cell JAG2expression influences endothelialcell tube formation. T47D cellswere precultured under hypoxiaand treated with control siRNA orsiRNA targeting JAG2. Hypoxia-treated T47D cells were thencocultured with MS1 endothelialcells. A, one representative light-microscopy image of coculturetube formation under differentT47D siRNA treatments. B,number of tubes formed in eachcoculture, expressed as relativenumber compared with non-siRNA–treated cocultures. Errorbars represent SD of the meanvalues from 3 independentexperiments. C, length of tubesformed in each coculture,expressed as relative numbercompared with non-siRNA–treated cocultures. Error barsrepresent SD of the mean valuesfrom 3 independent experiments.D, number of tubes formed in eachMS1 culture with mediaconditioned by hypoxic T47D cellsovernight as indicated. Error barsrepresent SD of the mean valuesfrom 3 independent experiments.*, indicates P < 0.05, in a one-wayANOVA (P ¼ 0.007) followed by aTukey HSD test.

Pietras et al.





suggested that branching of new vasculature is a result ofmodulation of Notch signaling within the endothelial cells.Activation of endothelial cells via secreted VEGF stimula-tion results in upregulation ofDLL4, which in turn activatesNotch in adjacent cells in a ligand-dependent manner (7).The VEGF stimulated DLL4-high endothelial cell, termedthe "tip-cell", direct the branching along the VEGF gra-dient. Meanwhile, the adjacent Notch-activated cells,termed the "stalk-cells", become less responsive to VEGFvia downregulation of VEGF-receptors, and instead take onthe proliferative role driving new vascular formation (7). Inaddition, stalk-cells also upregulate the Notch ligand JAG1that, in contrast to the Notch activating role of DLL4,results in inhibition of Notch-activation back on the tip-cell(42). In total, this results in a mechanism in which tip-cellswith low Notch-activity drive the branching of new vascu-lature, whereas stalk-cells with high Notch-activity drive thelengthening of the vessels. This mechanism is supported bymultiple studies in which inhibition of Notch signalingduring vascular development results in a hyper-branchingphenotype (38–41, 43).Considering the known involvement on Notch in the

process of angiogenesis, we speculated that hypoxic induc-tion of JAG2 and its influence on Notch signaling couldrepresent a novel mechanism for tumor-induced vasculo-genesis. Upon conducting a GO analysis on primary breastand RCC tumors, highly significant correlations betweenJAG2 expression and genes involved in angiogenic processeswere detected. This was further confirmed by performing aGSEA on complete JAG2 correlated ranked lists, againdisplaying significant enrichment for vascular develop-ment and angiogenesis gene signatures. In addition, cocul-ture experiments using hypoxic breast cancer epithelialcells and murine endothelial cells displayed significantlyreduced endothelial tube formation as was measured byboth number of branches and length of tubes. Interest-ingly, it has previously been shown in head and necksquamous cell carcinoma that growth factor–inducedJAG1 expression on tumor cells resulted in increased

tumor vascularization (35). It was hypothesized that thisincrease in angiogenic processes was a result of an increasein endothelial Notch-activity due to JAG1 stimulation.However, in light of recent work displaying an inhibitoryrole of JAG1 on Notch-activity in endothelial cells (42), itseems probable that the increased vascular branchingcould be attributed to Notch-inhibition as apposed toactivation. In line with this model, our coculture experi-ments also suggest vascular branching due to alterations toendothelial Notch activation via JAG2 stimulation, whichaccording to what is known today, is likely to be a Notch-inhibiting effect. Our findings bring to light the possibilityof local tumor cell–endothelial cell activating cues func-tioning in a juxtacrine manner that would contribute totumor endothelial cell angiogenesis in addition to theparacrine stimulation via VEGF.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Elise Nilsson and Siv Beckman for skillful technical assistanceand Dr. Kristian Pietras (Karolinska Institutet, Stockholm, Sweden) for providing theMS1 cells. The funders of the study had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Grant Support

This work was supported by the Swedish Cancer Society, the Children CancerFoundation of Sweden, the Swedish Research Council, the Swedish Foundationfor Strategic Research-supported Strategic Center for Translational CancerResearch, CREATE Health, the Strategic Cancer Research Program, BioCARE,the Ollie and Elof Ericsson Foundation, the Crafoord Foundation, the GunnarNilsson Foundation, and the research funds of Malm€o University Hospital.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 11, 2010; revised March 2, 2011; accepted March 3, 2011;published OnlineFirst March 14, 2011.

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2011;9:626-636. Published OnlineFirst March 14, 2011.Mol Cancer Res Alexander Pietras, Kristoffer von Stedingk, David Lindgren, et al. Signaling and Enhances Endothelial Cell Tube FormationJAG2 Induction in Hypoxic Tumor Cells Alters Notch

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