Expression of the Ets-1 Transcription Factor in Human ...Slides were then dehydrated in graded alcohols. 35S-labeled sense and anti-sense riboprobes were synthesized by in vitro transcription

[CANCER RESEARCH 59, 5608–5614, November 1, 1999]

Expression of the Ets-1 Transcription Factor in Human Astrocytomas Is Associatedwith Fms-like Tyrosine Kinase-1 (Flt-1)/Vascular Endothelial Growth FactorReceptor-1 Synthesis and Neoangiogenesis1

Markus M. Valter, 2 Anja Hugel, H-J. Su Huang, Webster K. Cavenee, Otmar D. Wiestler, Torsten Pietsch,3 andNicolas Wernert3

University of Bonn Medical Center, Departments of Neuropathology [M. M. V., O. D. W., T. P.] and Pathology [A. H., N. W.], D-53105 Bonn, Germany; Laboratory of TumorBiology, Ludwig Institute for Cancer Research and Department of Medicine, University of California-San Diego, La Jolla, California [H. S. H., W. K. C.]

ABSTRACT

Marked neovascularization and vascular endothelial proliferation are charac-teristic features of malignant gliomas. Vascular endothelial growth factor(VEGF), an angiogenic protein secreted by glioma cells, appears to play a crucialrole for induction of neoangiogenesis. The VEGF receptors fms-like tyrosinekinase-1 (Flt-1)/VEGFR-1 and kinase insert domain-containing receptor (KDR)/VEGFR-2 are up-regulated on the surface of endothelial cells (ECs) in gliomas.Both receptor genes contain an Ets-responsible element in their promoters. Theproto-oncogeneets-1encodes a transcription factor that has been associated withblood vessel formationin vivo under physiological and pathophysiological condi-tions including tumor neovascularization. Ets-1 is induced by VEGF in culturedECs. In vitro data also point to a role of Ets-1 as a transcriptional activator ofFlt-1. These properties prompted us to investigate Ets-1 expression in 32 humanastroglial tumors of WHO grades I-IV and to correlate the data with the expres-sion pattern of VEGF, Flt-1, and KDR. By in situ hybridization, high ets-1mRNAlevels were found in the glioma microvasculature with particularly prominentsignals in glomeruloid vascular endothelial proliferations of glioblastomas (WHOgrade IV). Semiquantitative reverse transcription-PCR identified the full-lengthets-1transcript but none of three known splice variants encoding isoforms withdifferent functional domains. Immunohistochemical staining demonstrated Ets-1protein preferentially in the nucleus of those ECs with an epithelioid morphologyconsistent with an activated state, whereas quiescent flat-shaped ECs predomi-nantly displayed cytosolic immunoreactivity. This observation proposes nucleartranslocation of Ets-1 during neoangiogenesis. VEGF synthesis by glioma cellswas accompanied by Ets-1 expression in adjacent microvascular ECs. Further-more, a highly significant correlation was observed between Ets-1 and Flt-1 (butnot KDR) expression in ECs of the glioma microvasculature. Our data suggestthat VEGF secreted by glioma cells induces Ets-1 in adjacent microvascular ECs,which subsequently transactivates the VEGF receptor Flt-1. This cascade maycrucially promote neoangiogenesis in human gliomas.

INTRODUCTION

Angiogenesis is of crucial importance for the development andgrowth of solid neoplasms. Some of the mechanisms that regulatetumor vascularization have been identified recently (for recent re-views, see Refs. 1, 2). VEGF4/vascular permeability factor, an angio-

genic protein that induces endothelial proliferation, serves as animportant modulator of neoangiogenesis in various tumors, includinghuman gliomas (3–5). VEGF and its endothelial high-affinity recep-tors Flt-1/VEGFR-1 (6) and KDR/Flk-1/VEGFR-2 (7) play a criticalrole during the development of mouse embryos (8–11) and are ex-pressed in the developing central nervous system. In normal adultbrain tissue, both VEGF and the two receptors appear significantlydown-regulated (for recent review see Refs. 12, 13). A strong induc-tion of VEGF transcripts and protein can be observed in astrocyticgliomas (14, 15), with concomitant expression of the VEGFRs Flt-1and KDR on tumor ECs (16). Flt-1 and KDR represent receptortyrosine kinases, which stimulate the Ras-Raf-mitogen-activated pro-tein kinase pathway and activate the transcription of genes involved intriggering the angiogenic program (17–21). Among the phosphoryla-tion targets of mitogen-activated protein kinase in ECs, the transcrip-tion factor Ets-1 has been identified (22–25). Interestingly, Ets-1 isinduced by VEGF in cultured ECs (26, 27), andin vivo expression ofEts-1 has been associated with new blood vessel formation under bothphysiological and pathophysiological conditions,e.g.,during embry-onal development, formation of granulation tissue, and tumor neovas-cularization (28–31). Ets-1 is also induced in the central nervoussystem of murine embryos but is down-regulated in the normal adultbrain (32).

The proto-oncogenec-ets-1is the cellular homologue of v-ets, theoncogene of avian acute leukosis retrovirus E26. It encodes theprototypic member of a novel family of transcription factors, the Etsproteins (33, 34; for a review see Refs. 35, 36). These proteins sharea common DNA-binding motif, the Ets domain, through which theyconvey transcriptional activation to promoters with an Ets-responsiveelement (24). Ets-1 has been implicated in both tumor invasion andneovascularization (27, 30, 31, 37); it up-regulates proteinases such asuPA, collagenase-1 (MMP-1), and stromelysin-1 (MMP-3), all ofwhich carry Ets-responsive elements in their promoters (27, 38).These proteinases degrade ExCM, a process during ExCM remodelingthat appears to be necessary for tumor invasion and neoangiogenesis.Strikingly, ets-1 antisense oligonucleotides abrogate the invadingphenotype, uPA and MMP-1 expression, and VEGF-induced migra-tion of ECs in vitro (26, 27). The gene for the EC-specific Flt-1/VEGFR-1 also contains an Ets-responsive element in its promoter,and mutational disruption of this Ets-1 binding site results in adecrease of Flt-1 transcription by 90%; moreover, Ets-1 transactivatesa Flt-1 promoter-reporter gene fusion construct in cultured ECs (39).By sequence analysis, we were also able to identify a putative Ets-responsive element in the KDR/VEGFR-2 gene promoter.

These properties raise the possibility that Ets-1 may be involved inthe regulation of VEGF-mediated tumor angiogenesis. To investigatea potential role of the Ets-1 transcription factor in neovascularizationof human gliomasin vivo, we studied the expression of Ets-1, VEGF,Flt-1, and KDR as well as the pattern of tumor vascularization in 32astrocytic tumors, including 8 pilocytic astrocytomas WHO grade I(A-I), 7 low-grade diffuse astrocytomas WHO grade II (A-II), 9

Received 5/3/99; accepted 9/3/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Deutsche Krebshilfe (Dr. Mildred Scheel Stiftung fu¨rKrebsforschung) and DFG (Deutsche Forschungsgemeinschaft; SFB 400). M. M. V. is afellow of the German National Scholarship Foundation.

2 Current address: Harvard University, Department of Molecular and Cellular Biology,Cambridge, Massachusetts 02138.

3 To whom requests for reprints should be addressed, at Departments of Pathology/Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25,D-53105 Bonn, Germany. E-mail: [email protected] (N. W.); E-mail:[email protected] (T. P.).

4 The abbreviations used are: VEGF(R), vascular endothelial growth factor (receptor);Flt-1, fms-like tyrosine kinase-1; KDR, kinase insert domain-containing receptor; Flk-1,fetal liver kinase-1 (murine homologue of human KDR); EC, endothelial cell; uPA,urokinase-type plasminogen activator; MMP, matrix metalloproteinase; ExCM, extracel-lular matrix; A-I, pilocytic astrocytoma WHO grade I; A-II, low-grade diffuse astrocy-toma WHO grade II; A-III, anaplastic astrocytoma WHO grade III; GBM-IV, glioblas-toma multiforme WHO grade IV; RT-PCR, reverse transcription-PCR; CD, cluster ofdifferentiation.

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anaplastic astrocytomas WHO grade III (A-III), and 8 cases of glio-blastoma multiforme WHO grade IV (GBM-IV).

MATERIALS AND METHODS

A total of 32 astrocytoma and 6 adult normal brain tissue specimens weretaken from the files and frozen tissue bank at the Department of Neuropathol-ogy, University of Bonn Medical Center, reviewed independently by twoneuropathologists, and classified according to the revised WHO grade classi-fication of brain tumors (40). The samples were processed for analysis imme-diately after surgery. For immunohistochemistry andin situ hybridization,specimens were fixed in 4% buffered formalin for 8 h and routinely processedfor paraffin embedding. Sections were cut at 4mm, mounted on positivelycharged slides (Superfrost1; Merck, Darmstadt, Germany) and air-dried in anincubator at 42°C overnight. For RNA and protein extraction, native tumorsamples had been snap frozen in liquid nitrogen and stored at280°C.

Frozen sections of native tumor samples were examined microscopically toexclude “contamination” with nonneoplastic or necrotic tissue. One hundred-fifty cryostat tumor sections (5mm 3 ;50 mm2) were harvested in liquidnitrogen and processed for either total RNA extracts or protein lysates. ForRNA extraction, samples were lysed in 500ml of TRIzol (Life Technologies),followed by the manufacturer’s protocol for RNA isolation and DNase Idigestion. Protein extracts were prepared by lysis for 30 min at 4°C in 500mlof buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% NP40, 1 mMphenylmethylsulfonyl fluoride, 10mg/ml leupeptin (Boehringer), and 100units/ml aprotinin (Calbiochem). Debris was removed by centrifugation for 10min at 13,0003 g and 4°C.

In Situ Hybridization. Prior to hybridization, sections were deparaffinizedin xylene and rehydrated in graded alcohols. They were subjected to gentleproteinase K digestion [1mg/ml in 0.1 M Tris-HCl (pH 8.0), 0.05M EDTA,0.1% SDS] for 10 min at 37°C, subsequent postfixation in 4% paraformalde-hyde for 15 min at room temperature, and acetylation with 0.25% acetic acidanhydride in 0.1M triethanolamine (pH 8.0) for 10 min at room temperature.Slides were then dehydrated in graded alcohols.35S-labeled sense and anti-sense riboprobes were synthesized byin vitro transcription (SP6 polymerasefor 1 h at37°C) from the respective humanets-1cDNA template (nucleotides260-1086, inserted into a pSP64 plasmid vector). DNase I digestion (1 U/ml)for 15 min at 37°C was followed by RNA isolation (TRIzol, manufacturer’sprotocol) and Sephadex G-50 column (Boehringer) purification. Hybridizationwas carried out for 16 h at 53°C [riboprobe activity, 23 104 cpm/ml;hybridization cocktail: 20 mM Tris-HCl (pH 8.0), 0.3M NaCl, 5 mM EDTA, 0.1M DTT, 0.5 mg/mlEscherichia colitRNA, 0.2 mg/ml Ficoll 400, 0.2 mg/mlpolyvinylpyrrolidone, 0.2 mg/ml BSA, 10% dextran sulfate, 50% deionizedformamide]. Unbound riboprobe was removed by stringent washing [20 mM

Tris-HCl (pH 8.0), 0.15M NaCl, 5 mM EDTA, 0.1 M DTT, 50% deionizedformamide) for 30 min at 64°C and RNase A digestion (0.02 mg/ml) for 45min at 37°C. After dehydration in graded alcohols, positive signals werevisualized by autoradiography (10-day exposure; photoemulsion NTB2;Kodak) and darkfield illumination of the slides following fluorescent nuclearcounterstaining (Hoechst 33258).

RT-PCR. Purified total RNA was reverse transcribed into cDNA using theSuperScript II RT kit (Life Technologies) primed with oligo(dT)12–18. To detectdifferent ets-1 mRNA isoforms, i.e., full-length transcript and splice variantslacking exon 4 and/or exon 7, RT-PCR was performed either with a primer pairflanking exon 4 (59-cgatctcaagccgactctca-39 and 59-gtcttagggcgatatggagc-39) orwith a primer pair flanking exon 7 (59-ccccagacaacatgtgcatg-39 and 59-tactcttt-gactcggcaccg-39). Depending on the presence or absence of the exon, amplifiedcDNAs theoretically result in products of 253 or 454 bp for exon 4, and 221 or 482bp for exon 7. Primers forb-actin (59-agccatgtatgtggccatcc-39 and 59-ggactgact-gatggagtact-39) were used to obtain a 182-bp standard amplicon for semiquanti-tative differential RT-PCR. Products were separated by gel electrophoresis inagarose and stained with ethidium bromide or, alternatively, detected by silverstaining in 8% acrylamide gels under denaturing conditions.

Immunohistochemistry. Sections were deparaffinized in xylene and rehy-drated in graded alcohols. For Flt-1 and KDR staining, specimens weresubjected to protease-induced epitope retrieval by incubation with 0.05%Pronase E (Sigma Chemical Co.) at 37°C for 5 and 2 min, respectively. ForEts-1 detection, microwave pretreatment [400W for 7 min in 10 mM sodium

citrate buffer (pH 6.0)] was used. Serial tumor sections were chosen forcoexpression studies of Ets-1 with Flt-1, KDR, VEGF, or CD34. Slides wereexposed to 1% hydrogen peroxide diluted in methanol for 30 min at roomtemperature to block endogenous peroxidase activity, followed by rehydrationin PBS. They were then incubated in a blocking solution (PBS containing 5%nonfat dry milk and 2% normal rabbit serum) for 30 min at room temperatureand twice for 15 min with an avidin/biotin blocking kit (Vector). The solutionwas removed from the slides by a filter paper, and the respective primaryantibody, diluted in 0.1% BSA-PBS, was added to the sections overnight at4°C. After unbound antibody was removed by several rinses with 0.1% TritonX-100 in PBS, the remaining specifically bound antibody was detected usingthe ABC method (DAKO) and visualized by diaminobenzidine tetrahydrochlo-ride. Sections were lightly counterstained with hematoxylin and analyzed bystandard light microscopy.

The following primary antibodies were used: anti-Ets-1 rabbit polyclonal serum(1:500; Santa Cruz), anti-CD34 monoclonal antibody (clone QBEND10, IgG1;Immunotech, Hamburg, Germany), anti-VEGF monoclonal antibody G153-694(IgG2b, 0.5mg/ml; Ref. 14); anti-Flt-1 polyclonal rabbit serum (1:75; Santa Cruz),and anti-KDR/Flk-1 polyclonal rabbit serum (1:250; Santa Cruz).

Western Blot Analysis. Cell lysates with 15–18mg of total protein wereseparated by electrophoresis on a 15% SDS-polyacrylamide gel (SDS-PAGE)and blotted onto a nitrocellulose membrane (Bio-Rad). After blocking with 5%nonfat dry milk in PBS for 2 h atroom temperature, filters were incubated withanti-Ets-1 rabbit polyclonal serum (1:500; Santa Cruz). Binding of the primaryantibody was detected by chemiluminescence with a horseradish peroxidase-conjugated secondary antibody and the SuperSignal substrate (Pierce).

Statistical Analysis. Statistical analysis was carried out by Fisher’s exacttest. Only correlations withP , 0.05 were accepted as significant.

RESULTS

Expression of ets-1 mRNA in Human Gliomas. To assess apotential role of Ets-1 as a modulator of tumor angiogenesis in humangliomas, the distribution and cellular localization ofets-1mRNA wasdetermined byin situ hybridization. High levels ofets-1 transcriptswere detected in ECs of malignant gliomas, and their distributioncorrelated with the extent of vascular endothelial proliferation. Astrongets-1mRNA signal could be observed in the microvasculatureof GBM-IV (seven of eight cases; Table 1 and Fig. 1,c andd), and inanaplastic astrocytomas WHO grade III (five of nine cases; Table 1).In GBM-IV, ets-1expression was particularly prominent in festoon-like vascular structures considered to represent sites of active neoan-giogenesis (40). Markedets-1labeling was also present in the vascu-lature of pilocytic astrocytomas WHO grade I, an astrocytoma entitywith frequent endothelial proliferation (six of eight; Table 1; Fig. 1,aand b). In contrast, low-grade diffuse astrocytomas, WHO grade II,without significant vascular proliferates did not usually exhibit endo-thelial ets-1 induction (Table 1; Fig. 1,e and f). The hybridizationsignal in this entity was similar to background levels observed in

Table 1 Expression of Ets-1, VEGF, Flt-1, and KDR in human astrocytomas

Eight pilocytic astrocytomas WHO grade I (A-I), seven low-grade diffuse astrocyto-mas WHO grade II (A-II), nine anaplastic astrocytomas WHO grade III (A-III), and eightglioblastomas WHO grade IV (GBM-IV) were analyzed separately for Ets-1 expressionand synthesis of VEGF, Flt-1, and KDR by immunohistochemistry.

VEGF1 VEGF2 Flt-11 Flt-12 KDR1 KDR2

A-I (n 5 8)Ets-11 50% 25% 75% 0% 37.5% 37.5%Ets-12 12.5% 12.5% 0% 25% 25% 0%

A-II (n 5 7)Ets-11 14.3% 0% 14.3% 0% 14.3% 0%Ets-12 28.6% 57.1% 14.3% 71.4% 71.4% 14.3%

A-III (n 5 9)Ets-11 55.6% 0% 55.5% 0% 55.6% 0%Ets-12 0% 44.4% 0% 44.4% 33.3% 11.1%

GBM-IV (n 5 8)Ets-11 87.5% 0% 87.5% 0% 62.5% 25%Ets-12 12.5% 0% 0% 12.5% 12.5% 0%

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ETS-1 EXPRESSION AND NEOANGIOGENESIS IN HUMAN GLIOMAS



normal adult brain tissue and in control hybridizations withets-1sense riboprobe (data not shown).

Gliomas Express Full-Lengthets-1mRNA. Alternative splicingof exons 4 and 7 results in isoforms ofets-1mRNA (41, 42) thatencode proteins with different functional domains (24, 36, 43). To

characterizeets-1 transcripts in astrocytic gliomas, semiquantitativedifferential RT-PCR was performed in representative samples foreach WHO grade (three A-I, two A-II, two A-III, and three GBM-IV).With primer pairs flanking either exon 4 or exon 7, only the longeramplicons including the respective exon could be detected (Fig. 2).

Fig. 1. Expression ofets-1mRNA and protein in human gliomas.a, c, ande, in situ hybridization analysis ofets-1mRNA. b, d, andf, corresponding H&E stains.a andb, pilocyticastrocytoma with prominentets-1 transcripts in microvascular structures;c and d, glioblastoma multiforme with prominentets-1 transcripts in microvascular structures.e and f, alow-grade diffuse astrocytoma WHO grade II without vascular endothelial proliferation lacks a specificets-1mRNA hybridization signal.g andh, immunohistochemical detection ofEts-1 protein. Anaplastic astrocytoma (g) and glioblastoma multiforme (h) exhibit immunoreactivity for Ets-1 protein in tumor capillaries.Scale bar, 100mm.

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This indicates that only the full-lengthets-1transcript is expressed inhuman gliomas. A good correlation between the levels of semiquan-titative RT-PCR products andin situ hybridization signals was foundin all cases. Normal adult brain tissue did not contain detectableamounts ofets-1mRNA.

Immunohistochemical Detection and Differential SubcellularLocalization of Ets-1 Protein. The amounts and cellular distributionof Ets-1 protein were examined by both immunoblotting and immu-nohistochemistry. The specificity of the commercial anti-Ets-1 poly-clonal rabbit serum was verified by immunoblotting analysis of celllysates from RSV-transfected 3T3 fibroblasts expressing the full-length Ets-1 p51; normal adult brain tissue revealed no Ets-1 signal(data not shown). All tumors with a positivein situ hybridizationsignal displayed a positive immunohistochemical reaction for thecorresponding Ets-1 protein. On the other hand, no Ets-1 protein couldbe demonstrated in gliomas without detectable transcripts. Normaladult brain tissue showed no Ets-1 immunoreactivity. Similar to thedistribution of mRNA, Ets-1 protein was expressed in ECs of thetumor microvasculature, particularly in festoon-like vascular struc-tures of GBM-IV (Fig. 1h), yet also in A-III (Fig. 1g) and A-I. ECs inlarger vessels were, if at all, only slightly stained. Expression of Ets-1significantly correlated with vascular endothelial proliferation as iden-tified by the endothelial CD34 surface antigen (P 5 0.0008; Fig. 3,aandb). In ECs with epithelioid morphology resembling activated ECs,Ets-1 protein appeared preferentially localized to the cell nucleus (Fig.3d). Other ECs displayed cytosolic Ets-1 immunoreactivity, and thesecells exhibited a flat morphology rather than an activated epithelioidphenotype. Benign A-II tumors, being rather poorly vascularizedwithout typical vascular proliferations (40), were Ets-1 negative withthe exception of a single tumor (Table 1).

Chemiluminescent immunoblotting analysis failed to demonstratedetectable levels of Ets-1 protein. This probably relates to the smallproportion of ECs present in total tissue extracts.

Complementary Expression of VEGF and Ets-1 in Gliomas.VEGF, a pivotal angiogenic factor, increases the transcription ofets-1in cultured human ECs (26, 27). Thus, VEGF constitutes an intriguingcandidate for an Ets-1-inducing signal in human gliomasin vivo.Immunohistochemical reactions for VEGF and Ets-1 were carried outin serial tumor sections. The strongest expression and highest numberof VEGF immunoreactive glioma cells were found in GBM-IV.Marked immunoreactivity could also be observed in A-III (Fig. 3c)and A-I, whereas it was less prominent in A-II (Table 1); no immu-noreactivity was seen in normal adult brain tissue. Some of thesefindings have been reported earlier (14). A significant correlation wasnoted between VEGF synthesis in glioma cells and Ets-1 expressionin juxtaposed microvascular ECs (P5 0.0009; Table 2; Fig. 3,c andd). This pattern would be consistent with a role for glioma-derivedVEGF as an inducer of Ets-1 in adjacent ECs.

Ets-1 Expression in Microvascular ECs Correlates with theDistribution of Flt-1/VEGFR-1. The genes for both VEGFRs, Flt-1/VEGFR-1 and KDR/VEGFR-2, contain a putative Ets-responsive ele-ment in their promoters, rendering them potentially susceptible for tran-scriptional activation by Ets-1. Moreover, it was demonstrated that Ets-1can transactivate Flt-1in vitro (39). In agreement with previous findings(16), Flt-1 as well as KDR were present on the surface of ECs in gliomas(Tables 1 and 2; Fig. 3,f-h), whereas normal adult brain tissue wasimmunohistochemically negative for either receptor. Flt-1 immunoreac-tivity was particularly prominent in GBM-IV but was also marked inA-III and A-I, whereas it was much less pronounced in A-II. In contrastto Flt-1, KDR expression was lowest in A-I. Furthermore, serial sectionsshowed a highly significantin vivo correlation between immunohisto-chemical signals for Ets-1 and Flt-1 (P 5 0.000000058; Table 2; Fig. 3,e-g) in ECs of corresponding microvascular structures. However, therewas no obvious relationship between Ets-1 and KDR (P 5 0.2695; Table2), which in contrast to Ets-1 and Flt-1 was frequently detected in largervessels (Fig. 3h).

DISCUSSION

Neoangiogenesis is of critical importance for the growth and develop-ment of astrocytic gliomas. Recent evidence suggests a crucial functionof VEGF as an angiogenic agent. However, little is known about themolecular pathways underlying VEGF-mediated endothelial growth. Theproto-oncogenec-ets-1encodes a transcription factor that has been asso-ciated with angiogenesis under both physiological and pathophysiologi-cal conditions (see “Introduction”). In the present study, strong expres-sion of ets-1mRNA and Ets-1 protein is demonstrated in ECs of theglioma microvasculature, especially in glomeruloid vascular proliferatesof high grade astrocytic gliomas, yet also in A-I,i.e., pediatric gliomascharacterized by bipolar morphology of the tumor cells and by a prom-inent microvasculature (40). In contrast, low-grade diffuse WHO grade IIastrocytomas, which display low vessel density and lack endothelialproliferation (40), failed to elicit detectable amounts ofets-1mRNA andprotein, except for one case with an unusually high degree of vascular-ization. Levels of the Ets-1 transcription factor showed a significantcorrelation with the extent of vascular proliferation in astrocytic gliomas(P 5 0.0008). Moreover, in epithelioid ECs, generally viewed as themorphological correlate of activated ECs, prominent nuclear Ets-1 im-munoreactivity could be observed, whereas quiescent, flat-shaped ECspreferentially displayed cytosolic Ets-1 staining. This suggests nuclearaccumulation of the Ets-1 protein during active neoangiogenesis in hu-man gliomas. Whether this involves active nuclear translocalization ofEts-1, perhaps even dependent on the cell environment, is subject offurther investigations comprisingin vitro and in vivo experiments. AnEts-1 site essential for nuclear translocation previously has been mappedto amino acids 369–388 and 364–441 (44). Among differentc-ets-1mRNA isoforms,i.e., a full-length transcript and splice variants lackingexon 4 and/or exon 7, only the full-length mRNA corresponding to thep51 Ets-1 protein (45) was found to be expressed in human gliomas asdetermined by RT-PCR. Thus, all critical functional domains of the Ets-1transcription factor appear to be present, including the protein-proteininteraction domain partially encoded by exon 4 (43) and the DNA-binding domain encoded by exons 7, 8, and 9 (36). Through thesedomains, Ets-1 conveys transcriptional activation to target genes thatcarry an Ets-responsive element in their promoter sequences. Our find-ings strongly indicate that the transcription factor Ets-1 participates in theregulation of endothelial proliferation in pilocytic astrocytomas, anaplas-tic astrocytomas, and glioblastoma multiforme.

VEGF also appears to play a crucial role during neoangiogenesis ofmany tumor entities, including human astrocytomas (3, 5). Inhibitionof VEGF signaling by targeting the ligand or its receptors has been

Fig. 2. RT-PCR analysis of full-lengthets-1 transcripts in human gliomas.a and b,Lanes 1–3, A-I;Lanes 4and5, A-II; Lanes 6and7, A-III; Lanes 8–10, GBM-IV; Lane11, normal brain tissue. A semiquantitative differential RT-PCR reaction was carried outwith representative glioma samples WHO grades I through IV. Amplification of ab-actincDNA fragment as a semiquantitative internal PCR control generated a 182-bp band. Foramplification ofets-1cDNA splice variants either primers flanking exon IV (a) or primersflanking exon VII (b) were used. Only products specific for unspliced cDNA with sizesof 482 (a) and 454 (b) bp were detected.

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demonstrated to repress both neoangiogenesis and tumor growth(46–48). Its expression is induced in gliomas (14, 15) and may berelated to the patients’ clinical prognosis (49). Immunohistochemicalreactions in the present study revealed a significant correlation be-

tween up-regulation of Ets-1 in microvascular ECs and VEGF expres-sion by neighboring glioma cells (P5 0.0009; Table 2), suggestingthat one function of glioma-derived VEGF may involve paracrinestimulation of Ets-1 in adjacent ECs followed by activation of neo-

Fig. 3. Immunohistochemical reactions for Ets-1, CD34, VEGF, Flt-1, and KDR.a, anaplastic astrocytoma with immunoreactive Ets-1 protein in tumor endothelia;b, EC markerCD34 surface antigen in tumor endothelia.c andd, glioblastoma multiforme immunolabeled for VEGF protein (c) and Ets-1 (d; note the epithelioid EC morphology). The glioma cellsexpress high levels of VEGF (c). e and f, glioblastoma multiforme stained for Ets-1- (e) and Flt-1- (f) immunoreactive protein. Labeling can be observed in tumor endothelia.g andh, immunolabeling for Flt-1 (g) in an anaplastic astrocytoma and KDR (h) in a low-grade diffuse astrocytoma (note different diameters of Flt-1-positiveversusKDR-positive vessels).Scale bar, 100mm.

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angiogenesis. This model is supported by recentin vitro data demon-strating that Ets-1 is up-regulated in cultured ECs upon exposure toVEGF. In addition,ets-1antisense oligonucleotides abolish VEGF-induced EC migration (26, 27). It has also been proposed that Ets-1stimulates its own expressionin vitro (50, 51). To confirm andcharacterize such an intriguing relationship between VEGF and Ets-1,further in vitro studies will be required.

Interestingly, both VEGFRs Flt-1/VEGFR-1 and KDR/VEGFR-2carry a putative Ets-responsive element in their promoters. By immu-nohistochemical analysis, we observed a highly significant correlationbetween the expression pattern of Ets-1 and Flt-1 in ECs of the gliomamicrovasculature (P5 0.000000058; Table 2). This suggests thatEts-1 may indeed function as a transcriptional activator for the Flt-1receptor gene in human gliomasin vivo. Support for this notion isprovided by recentin vitro data demonstrating that mutational dis-ruption of the Ets-responsive element in the Flt-1 promoter decreasestranscription of Flt-1 in cultured ECs by 90%. Moreover, Ets-1transactivates a Flt-1 promoter/reporter gene-fusion constructin vitro(39). Thus, VEGF secreted by glioma cells may up-regulate its high-affinity receptor Flt-1 on adjacent ECs through induction of Ets-1.Such a cascade would also explain the recentin vitro observation thatVEGF exposure leads to higher levels of Flt-1 in cultured ECs (52).Although a significant correlation was not found between Ets-1 andKDR expression (P5 0.2695; Table 2), our data cannot excludeKDRas another gene transactivated by Ets-1 in gliomas. In fact, only aminority of the gliomas examined (15.6%) displayed Ets-1 expressionwithout simultaneous KDR induction. On the other hand, KDR up-regulation occurred in 34.3% of the gliomas in the absence of detect-able Ets-1 expression. This suggests that Ets-1-independent mecha-nisms must be critically involved in conferring KDR induction (53–55). Future studies will have to address this issue in detail.

A previous report by Plateet al.(16) described an association betweenFlt-1 and KDR expression and histopathological grade in gliomas. Flt-1/VEGFR-1 appeared to be present in low-grade as well as in high-gradetumors, whereas KDR/VEGFR-2 was detected mainly in high-gradegliomas. Indeed, our data also show that high-grade gliomas often coex-press both VEGFRs, whereas A-I tumors that display conspicuous vas-cular proliferation preferentially contain Flt-1. On the other hand, 62.5%of the A-I tumor specimens examined expressed KDR/VEGFR-2 in ourseries, and A-II tumors, which lack prominent vascular endothelial pro-liferates, were immunoreactive for KDR even more frequently than forFlt-1 (Table 1). This difference compared with previously published datamay be due to increased sensitivity of immunohistochemical stainingafter protease-induced epitope retrieval as used in the current report (see“Materials and Methods”).

There is in vitro evidence suggesting that VEGF-mediated KDRactivation, but neither VEGF- nor PlGF-mediated Flt-1 activation,transduces a mitotic signal in cultured ECs (56–58). However,in vivoapproaches revealed that both Flk-1 (the murine homologue of KDR)and Flt-1 knockout mice have a strongly impaired vasculature result-ing in embryonic lethality by days 8–10 of gestation. Whereas onlyfew, if any, ECs are present in KDR(2/2) knockout animals (11), theFlt-1(2/2) phenotype exhibits only small patches of poorly organizedEC conglomerates (10). KDR is considered to transduce an EC-specific proliferative signal required for generating ECs, whereas

Flt-1 appears to be required during later steps of efficient EC orga-nization/generation. In fact, recentin vitro data argue for an involve-ment of Flt-1 in migration and activation of ECs (19, 59, 60). To whatextent these functional differences relate to the roles of Flt-1 and KDRin glioma angiogenesis remains to be determined.

In vitro studies have demonstrated that Ets-1 transactivates ExCM-degrading proteases that carry an Ets-responsive element in theirpromoters. Prominent examples include uPA, MMP-1, and MMP-3(27, 38). Ets-1 expression correlates with up-regulation of theseproteases, new blood vessel formation, and tumor cell invasionin vivo(28, 30, 37, 61). According to recent reports, induction of uPA inhuman gliomas may play a critical role for tumor cell invasion, tumorprogression, and prognosis (62–64). It has been shown that uPA isstrongly up-regulated in vascular endothelial proliferations of malig-nant gliomas (65). The fact that we observed Ets-1 induction inneoangiogenically active ECs would be compatible with a function forEts-1 as a uPA-inducing transcription factor in human gliomasin vivo.Proteolytic degradation of ExCM components by Ets-1-transactivateduPA may promote tumor vascularization and support tumor cellinvasion, a characteristic property of astrocytomas.

In summary, these data demonstrate associated expression patternsof VEGF, the VEGFR Flt-1 and the Ets-1 transcription factor inastrocytic gliomas. We propose a model for neoangiogenesis in as-trocytomas that uses a regulatory cascade of glioma-derived VEGF,binding of VEGF to the Flt-1 receptor tyrosine kinase on tumorendothelia, induction of Ets-1 in responsive ECs, and subsequentexecution of the angiogenic program comprising VEGFR up-regula-tion and ExCM degradation. Detailed functional studies will be re-quired to confirm this regulatory mechanism and to further charac-terize the underlying molecular pathways.

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Table 2 Summary of Ets-1, VEGF, Flt-1, and KDR expression in all 32 astrocytomas examined and Fisher’s exact statistical analysis of the significance of the respectivecoexpression

Gliomas (n5 32) VEGF1 VEGF2 Flt-11 Flt-12 KDR1 KDR2

Ets-11 53.1% 6.3% 59.4% 0% 43.8% 15.6%Ets-12 12.5% 28.1% 3.1% 37.5% 34.4% 6.2%

Positive correlation:P , 0.001 Positive correlation:P , 0.0000001

No significant correlation:P 5 0.2695

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1999;59:5608-5614. Cancer Res Markus M. Valter, Anja Hügel, H-J. Su Huang, et al. Synthesis and Neoangiogenesis(Flt-1)/Vascular Endothelial Growth Factor Receptor-1Astrocytomas Is Associated with Fms-like Tyrosine Kinase-1 Expression of the Ets-1 Transcription Factor in Human

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Expression of the Ets-1 Transcription Factor in Human ...Slides were then dehydrated in graded alcohols. 35S-labeled sense and anti-sense riboprobes were synthesized by in vitro transcription