Autocrine Endothelin-3/Endothelin Receptor B Signaling Maintains Cellular and Molecular Properties of Glioblastoma Stem Cells

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Published OnlineFirst October 19, 2011; DOI: 10.1158/1541-7786.MCR-10-0563

Molecular Cancer Cancer Genes and Genomics Research

Autocrine Endothelin-3/Endothelin Receptor B Signaling Maintains Cellular and Molecular Properties of Glioblastoma Stem Cells

Yue Liu1, Fei Ye1,9, Kazunari Yamada1, Jonathan L. Tso1, Yibei Zhang1, David H. Nguyen1, Qinghua Dong1,10, Horacio Soto2, Jinny Choe1, Anna Dembo1, Hayley Wheeler1, Ascia Eskin3, Ingrid Schmid4, William H. Yong5,8, Paul S. Mischel5,8, Timothy F. Cloughesy6,8, Harley I. Kornblum7,8, Stanley F. Nelson3,8, Linda M. Liau2,8, and Cho-Lea Tso1,8

Abstract Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression proﬁling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence. Mol Cancer Res; 9(12); 1668–85. 2011 AACR.

Introduction characterized as a small subset of stem-like tumor cells capable of initiating and sustaining tumor growth when Glioblastoma (WHO grade IV) is the most common and grafted into mice (2–6). Although CD133/prominin, a most aggressive type of primary brain tumor in humans. It normal neural stem cell (NSC) marker, is not an obligatory remains virtually incurable despite extensive surgical excision marker for GSCs (6, 7), CD133 was first applied as a surface marker for isolation and enrichment of GSCs (2–5, 7–9). and postoperative adjuvant radiotherapy and chemotherapy þ (1). Glioblastoma stem cells (GSC) have been recently Tumors initiated from CD133 GSCs often recapitulate the isolated from glioblastoma tumors of patients and were histopathologic features of the patient tumors from which the cells were derived, indicating the ability to self-renew and reproduce the cellular heterogeneity found in human glio- – þ Authors' Affiliations: Departments of 1Surgery/Surgical Oncology, 2Neu- blastoma tumors (2, 4 6). Studies showed that CD133 rosurgery, 3Human Genetics, 4Medicine/Hematology-Oncology, 5Pathol- tumor-initiating cells possess marked resistance to radio- ogy and Laboratory Medicine, 6Neurology, and 7Psychiatry, David Geffen 8 chemotherapy (10, 11) and thus are now suggested to be School of Medicine; Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California; 9Department of Neurosur- responsible for posttreatment failure and tumor recurrence. gery, Tongji Hospital, Tongji Medical College, Huazhong University of The molecular profiles of GSCs revealed characteristics of Science and Technology, Wuhan, Hubei; and 10Cancer Institute, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China neuroectodermal-like cells, expressing both neural and mesenchymal developmental genes, and portraying an undif- Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). ferentiated, migratory, astrogliogenic, and chondrogenic phenotype (8). This suggests that a subset of GSCs may Corresponding Author: Cho-Lea Tso, Department of Surgery/Surgical Oncology, Factor Building, Rm 13-260, 10833 Le Conte Avenue, David inherit neural crest cell (NCC)-like developmental pathways Geffen School of Medicine at UCLA, Los Angeles, CA 90095. Phone: 310- to initiate a tumor. In particular, endothelin 3 (EDN3), a 825-1066; Fax: 310-825-6192; E-mail: [email protected] potent mitogen for NCC and its derived lineage precursor doi: 10.1158/1541-7786.MCR-10-0563 cells (12, 13), was identified as one of the top genes highly 2011 American Association for Cancer Research. expressed in tumorigenic GSCs (8). EDN3 is a member of

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the endothelin (EDN) family, which consists of a group of xenograft animals (8). In parallel, autologous glioblastoma vasoactive peptides referred to as EDN1, EDN2, and EDN3 cell lines were established by propagating cells in DMEM/ (14). EDNs are synthesized initially as inactive larger pre- Ham's F-12 media supplemented with 10% FBS. cursor molecules and then posttranslationally cleaved by EDN-converting enzyme (ECE1) to yield the biologically Fluorescence-activated cell sorting analyses and þ active 21-amino acid form (15). The effects of EDNs are purification of CD133 GSCs mediated by 2 distinct but highly homologous G-protein– A total of 2 105 to 5 105 dissociated cells from coupled receptors, EDN receptor A (EDNRA) and EDN glioblastoma tumor sphere cultures were stained with anti– receptor B (EDNRB), in an autocrine and paracrine manner CD133-APC (Miltenyi Biotech) for 20 minutes at 4C. The (16, 17). The EDNRA predominantly binds to EDN1 and analyses for CD133 positive cells were done on a FACSCa- EDN2 with similar affinities, and EDN3 with 1,000- to libur flow cytometer (Becton Dickinson) and 10,000 or 2,000-fold lower affinity, whereas EDNRB has similar more events were collected in each analysis. To purify þ affinities for all 3 isopeptides (18). Mutations in EDN3 or CD133 cells, dissociated glioblastomas sphere cultures or EDNRB can lead to abnormal development of the enteric enzyme digested tumor xenografts were immunostained nervous system (ENS) and melanocytes and are known to with anti-CD133 APC (allophycocyanin) under sterile con- þ account for the majority of patients with Waardenburg ditions. The CD133 and CD133 cells were sorted and syndrome (WS) type IV, who exhibit both pigmentation collected on a BD FACSAria II cell sorter at 70 psi using a and megacolon phenotypes (19, 20). EDN3 and EDNRB 70-mm nozzle. mRNA expression has been reported in fetal human enteric mesenchyme and NCCs (21), and EDN3/EDNRB signal- Quantitative reverse transcriptase PCR analysis ing is known to influence NCC proliferation, differentia- Total RNA was extracted using a RNeasy Kit (QIAGEN). tion, and migration during ENS development (22). The Two micrograms of RNA from each sample were transcribed expression of the EDN system genes has been shown in the to cDNA using a TaqMan RT Reagent Kit (Applied Bio- brain and in human glioblastoma (23, 24), as well as a broad systems). PCR was done using 5 mL cDNA equivalents to range of other types of human cancers (25). The role of the 100 ng total RNA and was carried out by using SYBR Green EDN-axis, especially in EDN1-axis, has been implicated in PCR Core Reagents (Applied Biosystems). After amplifica- promoting tumor progression. Blocking EDN receptors has tion, PCR products (5 mL) were electrophoresed on 2% been suggested as a novel strategy in cancer therapy (25). agarose gel. The primer sequences and expected size of In this study, we provide the first comprehensive analysis amplified PCR products are described in Supplementary of the expression and function of the EDN system in patient- Information. derived GSCs. We found an essential role of autocrine EDN3/EDNRB system in GSCs. Blocking either EDNRB Immunocytochemical, histopathologic, and function or EDN3 production leads to GSC apoptosis and immunohistochemical analysis loss of migration, self-renewal, and tumorigenesis. Further- GSCs were fixed in 4% paraformaldehyde and then singly more, genome-wide expression array analyses have elucidat- or doubly stained with anti-CD133 (1:100; Abcam), anti- ed molecular pathways and gene network connections to this nestin (1:200; Chemicon), or anti-EDN3 (1:200; Santa essential signaling for maintaining GSCs. This finding Cruz Biotech) antibodies. After washing, cells were incu- implicates that the EDN3/EDNRB signaling pathway may bated with rhodamine red–conjugated anti-mouse or Alexa serve a novel therapeutic target for the development of Fluor 488–conjugated anti-rabbit antibodies (Invitrogen). potentially more effective treatment protocols for preventing Nuclei were counterstained with Hoechst 33342 (Invitro- GSC-mediated tumor recurrence. gen). Histopathologic analyses were done on frozen section or paraffin slides stained with hematoxylin and eosin (H&E) Materials and Methods staining as per standard technique. Immunohistochemical staining was done on paraffin slides. Slides were subject to a Glioblastoma sphere culture 1-hour blocking step followed by the application of primary Glioblastoma tumor specimens were obtained from antibody or control antibody for 1 hour at room temper- patients who underwent surgery at Ronald Reagan UCLA ature. The following primary antibodies were used: CD133 Medical Center. All samples were collected under protocols (1:100; Abcam), EDN3 (1:100; Epitomics), EDN1 (1:250; approved by the UCLA Institutional Review Board. Disso- Abcam), EDNRB (1:100; Epitomics), and EDNRA (1:200; ciated tumor cells from fresh tumor tissue were cultured in a ENZO life Science). The immunodetection was done using serum-free media containing Dulbecco's modified Eagle's Vectastain ABC Standard Kit and Vector NovaRED (Vector medium (DMEM)/Ham's F-12 (Mediatech) supplemented Laboratories). with 20 ng/mL human recombinant epidermal growth factor (EGF; Sigma), 20 ng/mL basic fibroblast growth ELISA factor (FGF; Millipore), 10 ng/mL leukemia inhibitory To determine the amount of EDN3 released from GSCs, factor (LIF; Millipore), and 1 B27 without vitamin A dissociated GSCs were plated in 24-well plates in triplicate at (Invitrogen) and is designated as stem cell growth factor the density of 5 104 cells per mL per well. Four hours later, (SGF) media. The tumorigenicity of GSCs was verified in stem cell culture media was replaced with fresh SGF media

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with or without ECE1 inhibitor, plain media or serum- determined 72 hours after transfection by quantitative containing media. The respective conditioned media were reverse transcriptase PCR (qRT-PCR), using Power SYBR collected at various time points and were stored at 80C. Green Cells-to-CT Kit (Ambion). The amount of EDN3 peptides in condition media was determined by an ELISA Kit specific for EDN3 (Immuno- Intracranial tumor formation assay and histopathologic Biological Laboratories). The absorbance was read at 450 nm analysis by Epoch Microplate Reader (BioTek Instruments). The role of EDN3/EDNRB signaling in maintaining the tumorigenic capacity of GSCs was examined in Beige/severe Flow cytometric analysis for cell-cycle distribution and combined immunodeficient mice (SCID) mice. A total of cell apoptosis 104 live GSCs treated with and without 50 mmol/L BQ788 To determine the effects of BQ788, BQ123, or combi- for 24 hours were injected in a total volume of 3 mL into nation treatment on cell-cycle distribution and cell apopto- brains of mice under a UCLA Institutional Animal Research sis, 2 105 to 5 105 treated and untreated GSCs were Committee–approved protocol. Mice were maintained for stained with propidium iodide (PI)-based hypotonic DNA 25 weeks or until development of neurologic signs. Brains of staining buffer. Cell-cycle distribution for approximately euthanized mice were collected, fixed in 10% formalin, 10,000 cells was analyzed using a FACScan (Becton Dick- paraffin-embedded, and sectioned. Alternatively, brain tis- inson). Apoptotic cells with degraded DNA were detected as sue was placed in O.C.T. embedding medium (Tissue- a hypodiploid or "sub-G1" peak in a DNA histogram. Tekm), dipped in liquid nitrogen, and sectioned in a 20 C cryostat. Histopathologic analyses were done on frozen Proliferation assay for GSCs section or paraffin slides stained with H&E staining as per The effects of BQ788 and/or BQ123 treatment on the standard technique. proliferative activity of GSCs were determined by a MTS/ PMS (phenazine methosulfate) colorimetric assay according Microarray procedures, data analysis, and gene to the manufacturer's instructions (Promega). Cells were annotation seeded into 96-well tissue culture plates at a density of 2,000 Molecular profiling of 6 untreated (n ¼ 3, duplicate) and 3 cells per well in the presence or absence of EDNRA or BQ788-treated GSCs (n ¼ 3) were carried out using EDNRB antagonists and incubated for 72 hours. The standard Affymetrix protocols and hybridized to Affymetrix absorbance was measured at 490 nm after a 4-hour incu- GeneChip U133 Plus 2.0 Array as described previously (8). bation with MTS/PMS reagents. The DCP files were globally normalized, and gene expression values were generated using the dChip implementation of Quantitative measurement of cell apoptosis and cell perfect-match minus mismatch model-based expression viability index. The group comparisons were done in dChip and To determine whether EDN3 alone can support GSC samples were permuted to assess the false discovery rate survival, cell apoptosis assays were done by using a Cell (FDR). To avoid inclusion of low level and unreliable signals, Death Detection ELISAPLUS kits (Roche), which quanti- the difference of the means needed to exceed 100 and be tatively detects the amount of cleaved DNA/histone com- called present byMAS5.0 ingreaterthan20% ofthe samples. plexes (nucleosomes) in a given sample. The GSCs were In addition, the Gene Set Enrichment Analysis (GSEA) tool seeded at the concentration of 2,000 cells/well/100 mLin was done to identify significant gene ontology groups that triplicates. The culture media was changed and switched to were enriched (26). Functional annotation of individual gene plain media that contain various concentrations of EDN3. was obtained from NCBI/Entrez Gene (http://www.ncbi. Cell apoptosis was measured after 72 hours incubation by nlm.nih.gov/sites/entrez), UniProt (http://www.uniprot. reading the absorbance at 405 nm. The same method was org/), information hyperlinked over protein (http://www. applied to the measurement of BQ788 and EDN3siRNA ihop-net.org/), and the published literature in PubMed treatment-induced cell apoptosis. The viability of GSCs was Central (http://www.ncbi.nlm.nih.gov/pubmed). determined by Trypan Blue staining (Invitrogen). Statistical analysis Knockdown of EDN3 by siRNA transfection Each experiment was set up in triplicate and repeated at A reverse transfection protocol was done to deliver EDN3 least twice. Data were expressed as means SD and analyzed siRNA (Ambion), nonsilencing control siRNA (Ambion) using 1-way ANOVA tests, depending on homogeneity of or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) variances. All P values were 2-sided, and values less than 0.05 siRNA (Dharmacon) into GSCs. Briefly, a transfection were considered significant. SPSS v13.0 for Windows soft- complex was prepared by diluting siRNA in 10 mL ware was used for all statistical analysis. OPTI-MEMI (Invitrogen), then adding 10 mL OPTI- MEMI containing 0.3 mL Lipofectamine RNAiMAX trans- Results fection reagent (Invitrogen). This complex was then added into each well in 96-well plate followed by seeding 6,000 Expression of the EDN system components in GSCs GSCs in 100 mL SGF media to give a final siRNA concen- Through genome-wide expression microarray analysis, we tration of 30 nmol/L in each well. EDN3 gene silencing was previously identified EDN3 as one of the most overexpressed

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EDN3 is a Survival Factor for Glioblastoma Stem Cells

AB 0.1% D431 D431 D431 S496 S496 D431 D431 S496 S496 cell line cell line cell line cell line Anti–Ig γ 1-APC cells cells cells cells cells cells cells cells + – + – + – + –

39.6% D431 Cell line (+ 6 h SGF) Unsorted spheres Sorted CD133 Cell line (serum) Cell line (serum) Cell line (+ 6 h SGF) Unsorted spheres Sorted CD133 Fibroblast MW Cell line (serum) Cell line (+ 6 h SGF) Unsorted spheres Sorted CD133 Cell line (serum) Cell line (+ 6 h SGF) Unsorted spheres Sorted CD133 Fibroblast E445 cell line (serum) E445 cell line (+ 6 h SGF) E445 25 μm D431 Sorted CD133 Sorted CD133 Sorted CD133 Sorted CD133 EDN1 EDN3 -EDN3 (250 bp) (110 bp) 9.6% S496 -EDN1 -EDNRA EDNRB EDNRA -EDNRB S496 (197 bp) (119 bp) -ECE1 - β-Actin 1.5% E445 β ECE1 -Actin (152 bp) (184 bp) 1 200 bp E445 100 bp β Anti–CD133-APC Anti–CD133-APC Anti–CD133-APC -Actintitration 50 bp

Figure 1. Presence of EDN system components in GSCs. A, tumorigenic GSC cultures, D431, S496, and E445, were established from patient-derived þ glioblastoma tumors. Percentages of CD133 cells in each culture were determined by flow cytometry analysis using anti-CD133 directly conjugated to APC. GSC cultures form semiadherent tumor spheres with cells spontaneously migrating out of spheres. Scale bar ¼ 25 mm. B, total RNA from the indicated GSCs cultured in SGF media, autologous glioblastoma cell lines cultured in serum-containing media and switched to SGF media for 6 hours and fibroblasts cultured in serum-containing media were extracted. The mRNA expression levels of EDN1, EDN3, EDNRA, EDNRB, and ECE1 were analyzed by qRT-PCR with specific primers. b-Actin was used as an internal control gene. transcripts in tumorigenic GSCs, when compared with EDN1, is a molecular/cellularsignatureforGSCs. þ autologous, differentiated glioblastoma cells cultured in Although both CD133 and CD133 cells sorted from serum-containing media (8). We thus hypothesized that glioblastoma sphere cultures express EDN3, differentiated EDN3 may contribute to maintaining GSC properties. To CD133 glioblastoma cell lines cultured in serum do not test this hypothesis, we first investigated the expression of the express EDN3, indicating that EDN3 is only expressed in EDN system genes in GSC cultures. Three patient-derived GSCs and their immediately differentiated CD133 tumorigenic GSC lines (D431, S496, and E445), which daughter cells maintained in stem cell culture condition. þ contain 39.6%, 9.6%, and 1.5% CD133 cells, respective- The data also suggest that GSC lines represent different ly, were employed for this study (Fig. 1A). GSC, which form cell populations from autologous glioblastoma cell lines, semiadherent tumor spheres with highly motile cells, spon- which cannot be subsequently reversed following transi- taneously migrate outward from sphere bodies (Fig. 1A). We tion to SGF conditions (27). þ tested both unsorted and sorted CD133 and CD133 cells from D431 and S496 GSC cultures, as well as the autologous Upregulation of EDN3 mRNA coupled with CD133 glioblastoma cell lines passaged in serum-contain- downregulation of EDN1 mRNA expression ing media. Because E445 GSC cultures contain only 1.5% characterizes stemness state of GSCs þ CD133 cells, we only tested the unsorted cell population. EDN1 has been implicated in the growth and progres- By using qRT-PCR analysis, mRNA expression of EDN1, sion of a wide range of human tumors (25), whereas EDNRA, EDNRB, and ECE1 was detected in all glioblas- recently, EDN3 has been suggested as a tumor suppressor toma cell samples tested. Notably, the expressions level of gene in female malignances (28, 29). Because we detected EDN1 and EDNRA mRNAs were particularly upregulated a reciprocal relationship in the expression of EDN1 and in all glioblastoma cell lines cultured in 10% serum, whereas EDN3 mRNA in glioblastoma cells cultured under dif- high levels of EDN3 mRNA was only detected in GSC lines ferent conditions, we examined whether there is a recip- established in SGF media, unsorted GSCs, or sorted rocal regulation of EDN1 and EDN3 in GSCs. FBS, þ CD133 or CD133 cells (Fig. 1B). EDN3 mRNA seems which contains a variety of cytokines and growth factors, to not be a SGF responsive gene or direct target of SGF was used as a surrogate for inflammatory mediators to because switching serum-cultured glioblastoma cells to SGF force GSC cultures to undergo proliferative differentiation for 6, 24, and 48 hours (Fig. 1B, not shown) did not as transit-amplifying-like cells capable of proliferation and stimulate comparable EDN3 mRNA expression in glioblas- differentiation, which eventually can be propagated as toma cells (Fig. 1B). These data indicate that EDN3, but not glioblastoma cell lines (Fig. 2A). Progressively diminished

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A C Patient tumor # D431 S496 E445 Figure 2. Reciprocal regulation and 1 2 3 4 5 6 7 8 9 10 11 expression of EDN1 and EDN3 mRNA in GSCs and glioblastoma tumors. A, light-microscopic a c e 25 μm EDN3 (110 bp) morphology of indicated GSC 200 bp cultures before (a, c, e) and after 100 bp 50 bp 2-week serum-induced proliferative differentiation (b, d, f). Scale bar ¼ EDN1 (250 bp) b +serumd +serum f +serum 25 mm. B, the indicated GSC cultures were switched to serum- B D431 S496 E445 containing media for the time shown. The mRNA expression EDNRB (197 bp) levels of EDN1 and EDN3 and the indicated NSC (CD133, SOX2, ID4), RGC (FABP7), and glioblastoma- associated genes (YKL-40, TOP2A) D431 cell line S496 cell line SGF E445 cell line SGF +serum-1 wk +serum-2 wk SGF +serum-1 wk +serum-2 wk +serum-1 wk +serum-2 wk EDNRA (119 bp) were analyzed by qRT-PCR with -EDN3 speciﬁc primers. b-Actin and -EDN1 GAPDH were used as internal -β-Actin ECE1 (184 bp) control genes. C, total RNA from patient-derived glioblastoma -CD133 tumors (n ¼ 11) were extracted. The -FABP7 mRNA expression levels of the indicated EDN system components -ID4 YKL-40 (206 bp) were analyzed by qRT-PCR with -GAP43 speciﬁc primers. YKL-40 -SOX2 represents a glioblastoma progression-associated gene, and -YKL-40 ββ-Actin (152 bp) b-actin was used as an internal -TOP2A control gene. -GAPDH

EDN3 mRNA expression coupled with marked induction GSCs are actively secreting EDN3 peptides when of EDN1 mRNA was seen in serum-stimulated GSC maintained in SGF conditions cultures (Fig. 2B). Serum-induced GSC differentiation Because EDN3 mRNA is uniquely expressed in GSCs, was further characterized by the codownregulation of but not in more differentiated autologous glioblastoma cells several radial glial cells (RGC)- and NSC-associated genes cultured in the serum, we then tested whether EDN3 was expressed by GSCs (ref. 8; Fig. 2B), including fatty acid- expressed at the protein level and secreted by GSCs main- binding protein 7 (FABP7; ref. 30), CD133 (31), sex- tained in SGF media. Immunostaining analysis revealed that determining region Y-box 2 (SOX2; ref. 32), inhibitor of EDN3 was only expressed in GSCs, but not in autologous DNA binding 4 (ID4; ref. 33), and GAP43 (34), suggest- glioblastoma cells cultured in serum-containing media (Fig. þ ing that EDN3 is a marker for undifferentiated, stem-like 3A). Moreover, sorted CD133 GSCs coexpressed EDN3 glioblastoma cells. In contrast, the expressions of EDN1 and CD133 or nestin, whereas glioblastoma cell lines only þ (glioblastoma tumor marker; ref. 23) and YKL-40 mRNA expressed nestin, not EDN3 or CD133. Either CD133 or (glioblastoma prognostic marker; ref. 35, only expressed in CD133 cells in all 3 tested sphere cultures express EDN3 as D431 and S496), were induced as a result of proliferative determined by 2-color flow cytometric analysis (Supple- differentiation (Fig. 2B). The mRNA level of topoisom- mentary Fig. S1). ELISA assays further confirmed that the erase (DNA) II alpha (TOP2A), a maker that is associated conditioned media collected from GSC cultures contained with cell growth, was unchanged. This data supports the high concentration of EDN3, although no EDN3 peptides notion that the reciprocal regulation of EDN3 and EDN1 were measured in conditioned media collected from glio- expression in GSCs may modulate and determine tumor blastoma cell line cultures (Fig. 3B). The production of development (36, 37). Indeed, the transcripts of EDN EDN3 peptides by GSC cultures was markedly decreased system genes were determined in patient-derived glioblas- when cultures were switched to plain media or serum- toma tumors, and elevated expression of EDN1 mRNA containing media (1 week), or treated with the ECE1 was determined in all 11 tumors analyzed, whereas EDN3 inhibitor SM-19721, by which GSC were forced to undergo mRNA was expressed at a lower level in the majority of starvation, proliferative differentiation, and acute EDN3 tested primary tumors when compared with EDN1 deficiency (Fig. 3B). These data confirmed that bioactive mRNA (Fig. 2C). Likewise, YKL-40 mRNA was EDN3 peptides were only actively secreted by GSCs main- expressed in most glioblastoma tumors (Fig. 2C). tained in SGF media. Moreover, under light microscope,

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A Fibroblast D431 cell line Sorted CD133+ Sorted CD133+ Sorted CD133+ S496 cell line D431 D431 S496

CD133/nestin DAPI DAPI DAPI DAPI CD133/DAPI DAPI Sorted CD133+ D431

CD133/nestin EDN3 EDN3 CD133 EDN3 EDN3/DAPI DAPI Sorted CD133+ 25 μm S496

CD133/EDN3 CD133/nestin Merge Merge Merge Merge DAPI DAPI

600 D431 S496 E445 Fibroblast

500 25 μm 400 untreated untreated 300 a d untreated g untreated j 200

EDN3 (pg/mL) 100 0 10 μmol/L 10 μmol/L 10 μmol/L 10 μmol/L b SM-19721 e SM-19721 h SM-19721 k SM-19721 S496 (day 1) S496 (day 2) E445 (day 1) E445 (day 2) D431 (day 1) D431 (day 2) S496 cell line E445 cell line D431 cell line +plain media +plain media +plain media + 1 wk serum + 1 wk serum + 1 wk serum 25 μmol/L 25 μmol/L 25 μmol/L 25 μmol/L +ECE1 inhibitor + ECE1 inhibitor + ECE1 inhibitor Medium (no cells) c SM-19721 f SM-19721 i SM-19721 l SM-19721

Figure 3. GSCs express and secret EDN3. A, the indicated GSC lines and autologous glioblastoma cell lines were subjected to single or dual color-fluorescent immunocytochemical analysis for the expression of EDN3, CD133, and nestin. The fluorescent stain 40, 6-diamidino-2-phenylindole (DAPI) was used to counterstain nuclei. Scale bar ¼ 25 mm. B, the amounts of EDN3 protein peptides secreted into the conditioned media by GSCs (5 104 cells/mL/well) cultured under the indicated conditions were determined by an anti-EDN3 quantitative ELISA kit and were presented as picograms per milliliter (pg/mL). The indicated glioblastoma cell lines were cultured in serum-containing media. Data represent mean values SD of triplicate measurements in triplicate experiments. C, light-microscopic morphology of GSC cultures and fibroblasts (5 104 cells/mL/well) treated with and without ECE1 inhibitor (SM-1972) at the indicated concentrations for 7 days. Scale bar ¼ 25 mm. acute cell death was observed when GSCs were treated with Evidently, structural/morphologic alterations and cell apo- the ECE1 inhibitor (Fig. 3C), suggesting that autocrine ptosis were only detected in GSCs treated with BQ788, not EDN3 may be an essential survival factor only when GSCs BQ123 or vehicle control (ethanol), as examined micro- are maintained in SGF conditions, not when GSCs undergo scopically, and BQ788-induced morphologic changes and cell differentiation. cell apoptosis were significantly enhanced in a dose-dependent manner (Fig. 4A). Correspondingly, the increase in Blockade of EDNRB, not EDNRA, signaling induces apoptotic cells (2.5- to 4-fold) appeared in a sub-G1 peak in GSC apoptosis without prominent cell-cycle arrest DNA histograms analyzed by flow cytometric analysis and Because proteolytic processing of EDN precursors to was only found in GSCs treated with BQ788 (50 mmol/L), biologically active peptides by ECE1 is not specificto not BQ123 (50 mmol/L), in all 3 GSC cultures tested (Fig. EDN3, we then analyzed and compared the effects of 4B). Notably, no major changes were found in the cell-cycle EDNRB and EDNRA antagonists on GSCs. GSCs were distribution in BQ788-treated GSCs when compared with treated for 72 hours with various doses of the EDNRB untreated control cells. Only slightly increased numbers of antagonist BQ788 or the EDNRA antagonist BQ123. cells in G1-phase (11%), S-phase (8%), and G2/M phases

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A B D431 S496 E445 45.1% D431 D431 S496 60.3% E445 47.2% Untreated Untreated Untreated

afuntreated untreated k untreated 15.0% 42.0% 24.2% 46.5% 13.0% 6.1% 15.5% 5.8% Solvent 6.3% 50 μm p (alcohol) 25 μmol/L 25 μmol/L 25 μmol/L b BQ788 g BQ788 l BQ788 56.3% 48.2% 35.0% BQ788 BQ788 BQ788 40.3% 50 μmol/L 50 μmol/L 50 μmol/L 25.5% 21.3% 48.2% μ μ μ 29.4% 32.1% 19.7% c 50 mol/L h 50 mol/L m 50 mol/L 16.8% BQ788 BQ788 BQ788 100 μmol/L 14.4% q BQ123

61.1% 75 μmol/L 75 μmol/L 75 μmol/L 54.6% 48.2% d BQ788 i BQ788 n BQ788 BQ123 BQ123 50 μmol/L μ BQ123 50 mol/L 50 μmol/L 25 μm 13.3% 33.7% 23.4% 46.4% 11.7% 15.5% μ 6.7% 100 μmol/L 100 μmol/L 100 μmol/L 100 mol/L 6.1% 5.5% ejBQ788 BQ788 oBQ788 r BQ788

C 160 a BQ788 BQ123 140 120 100 80 60 %Growth 40 20 0 7550250 100 7550250 100 7550250 100 0 100755025 μm μm μm μm D431 Sorted CD133+D431 Sorted CD133- D431 D431 cell line

160 b BQ788 BQ123 140 120 100 80 60 %Growth 40 20 0 7550250 100 7550250 100 7550250 100 0 100755025 μm μm μm μm S496 Sorted CD133+S496 Sorted CD133-S496 S496 cell line

c 160 de160 BQ788 BQ123 BQ788 BQ123 140 140 160 untreated alcohol BQ123 BQ788 BQ123+BQ788 120 120 140 100 100 120 80 80 100 80

%Growth 60 %Growth 60 60 40 40 %Growth 40 20 20 20 0 0 0 0 755025 100 7550250 100 D431 S496 E445 μm μm E445 Fibroblast

Figure 4. Blockade of EDNRB not EDNRA induces GSC apoptosis independent from cell-cycle arrest. A, light-microscopic morphology of GSC cultures treated with the indicated concentrations of BQ788, BQ123, or vehicle control (ethanol alcohol) for 3 days. Scale bar ¼ 25 or 50 mm. B, the indicated GSC lines were treated with 50 mmol/L BQ788 or BQ123 for 3 days, and cell-cycle distributions including sub-G1 fraction were determined by ﬂow cytometric analysis. C, the sorted CD133þ and CD133 cells and unsorted GSC lines, autologous glioblastoma cell lines, and ﬁbroblasts were treated with BQ788, BQ123 (a–d), or combinations (e) at the indicated concentrations for 3 days. The cell growth was determined by MTS/PMS cell proliferation assays. Data represent mean values SD of triplicate measurements in triplicate experiments.

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and S496) are sensitive to BQ788 treatment. BQ788 treat- þ D ment also decreased clonogenic efficiency of both CD133 50 and CD133 daughter cells sorted from GSC cultures that þ 500 250 120 were initiated from purified CD133 GSCs (Fig. 4D). 40 (4.4 %) (1.7 %) (0.0 %) (0.0 %) (1.6 %) (0.0 %) (4.8 %) (0.8 %) (0.0 %) (0.0 %) 60 30 15 Notably, untreated CD133 daughter cells are fast-growing 30 cells, which exhibited higher clonogenic efficiency compared þ 0 %) 2 %) 4 %) 0 %) 0 %) 0 %) 8 %) 0 %) 5 %) 6 %) ...... 20 . with those of untreated CD133 daughter cells on day 7 (0 (1 (2 (0 (0 (0 (0 (2 (2. (8. after cell seeding. 10 Colony counts To verify that the observed decreased proliferative activity 0 and clonogenic efficiency caused by BQ788 is mainly due to Untreated 50 µmol/L BQ788 Untreated 50 µmol/L BQ788 cell apoptosis, not cell-cycle arrest, the induction of GSC sorted CD133+ cells sorted CD133- cells apoptosis by the EDNRB antagonist was further determined using an ELISA-based cell death assay, which quantitatively BQ788 0 µmol/L BQ788 50 µmol/L E BQ788 25 µmol/L BQ788 100 µmol/L detects the amount of cleaved DNA/histone complexes. 1 fi * * Signi cant cell apoptosis were detected in all 3 GSC lines 0.9 treated with 50 to 100 mmol/L BQ788 when compared with 0.8 * * untreated cells (Fig. 4E). Thus, cell morphology observa- 0.7 * 0.6 tions, clonogenic assays, cell-cycle analyses, and cell apopto- 0.5 * sis assays suggest that EDN3/EDNRB signaling is a survival bance 405 nm) bance 405 of cytoplasmic nucleosomes 0.4 pathway for GSCs. 0.3 oligo

(absor 0.2

Amount Blockade of EDN3/EDNRB signaling leads to the loss of 0.1 0 cell spreading/migration, self-renewal, and tumor D431 S496 E445 initiation Next, we determined whether blockade of EDN3/

þ EDNRB signaling would weaken GSC self-renewal capa- Figure 4. (Continued) D, CD133 and CD133 daughter cells sorted fi þ bility. Signi cant cell apoptosis and impaired GSC migra- from sphere cultures that initiated from purified CD133 GSCs were seeded at the indicated cell density per well in 96-well plate by limiting tion were detected in all 3 GSC lines treated with 50 mmol/L dilution in 100 mL SGF media. The clonogenic efficiency was determined BQ788 when compared with untreated cells (Fig. 5A, a–f). under the presence or absence of 50 mmol/L BQ788 treatment. Colonies More importantly, these BQ788-treated GSCs failed to containing more than 20 cells were counted at day 7 after cell seeding. regenerate spheres when live cells were reseeded at clonal Data represent mean values SD of triplicate measurements in triplicate experiments. % clonogenic efficiency was indicated. E, the indicated density (Fig. 5A, h, j, l), indicating that the self-renewal GSC cultures were treated with BQ788 at the indicated concentrations for capacity of GSCs was lost. The impairment of cell migration 3 days, and cell apoptosis were determined by a Cell Death Detection and self-renewal by BQ788 treatment was also shown in þ ELISAPLUS kit, which quantitatively detects the amount of cytoplasmic purified CD133 GSCs (Supplementary Fig. S2). To study oligonucleosomes. Data represent mean values SD of triplicate whether blocking the production of endogenous EDN3 in measurements in triplicate experiments. , P < 0.05 versus untreated cells. GSCs will have impact on these GSC functions, we carried out an on-target knockdown of EDN3. Treatment with EDN3 siRNA, not negative control siRNA, not only effi- (10%) were detected in D431, S496, and E445 GSCs, ciently knocked down EDN3 mRNA expression (Fig. 5B), respectively, after BQ788 treatment (Fig. 4B). but also abrogated the EDN3 peptide release (Fig. 5C). To gain further insight into the effects of the blockade of More importantly, prominent cell apoptosis was only deter- EDNRB on GSC growth, GSCs were treated for 72 hours mined in GSCs treated with EDN3 siRNA (Fig. 5D). with various doses of BQ788 or BQ123 (Fig. 4C) and Microscopic changes in cellular morphology were observed, assayed for cell proliferation. Inhibition of proliferation was and in contrast to negative control siRNA-treated GSCs not seen when GSCs (sorted or unsorted GSC) were treated (Fig. 5E, c, g, k), loss of endogenous EDN3 by EDN3 with 25 to 100 mmol/L BQ123, although treatment with 75 siRNA treatment resulted in loss of self-renewal potential or 100 mmol/L BQ788 reduced cell proliferation by 30% to and cell migration capacity (Fig. 5E, d, h, l). By contrast, 80% in D431, 30% to 60% in S496 and 40% to 60% in GSCs in negative control siRNA-treated cultures spontane- E445 (Fig. 4C, a–c). The combination of BQ788 and ously migrated/spread out of tumor spheres and formed the BQ123 had no additive effect on suppressing cell growth, surrounding monolayer (Fig. 5E, q–s), implicating a role of although a notable growth stimulation was observed in EDN3/EDNRB signaling in GSC self-renewal and unsorted D431 and S496 by treatment with 50 mmol/L migration. BQ123 (Fig. 4, e). Of note, both fibroblasts and autologous On the basis of the requirement of EDN3/EDNRB glioblastoma cell lines passaged in serum-containing media signaling for self-renewal, migration, and survival of GSCs that do not produce EDN3 were resistant to BQ788 in culture, we test whether the EDN3/EDNRB signaling is þ treatment (Fig. 4C, a–d). In contrast, both CD133 and required for maintaining the tumorigenic potential of þ CD133 daughter cells sorted from sphere culture (D431 GSCs. Unsorted GSCs or sorted CD133 GSCs were

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treated with or without 50 mmol/L BQ788 for 24 hours. Exogenous EDN3 alone can maintain GSC survival but The viability of the inhibitor-treated cells, which ranged not promote GSC expansion from 80% to 87%, was determined by trypan blue staining. To explore the possibility that EDN3 alone might be a Following washing, 104 live cells were stereotactically growth factor or a survival factor for GSCs, we removed stem injected into the brains of SCID mice. Mice (15/16) which cell growth factors (FGF, EGF, LIF) from the media and received untreated GSCs developed neurologic signs at replaced them with various doses of recombinant EDN3. weeks 14 to 22 postinjection. The H&E staining of tumor Apoptotic cells were observed in all tested GSCs maintained xenografts identified histologic hallmarks of human glio- in plain media (40%–50% viable cells) and the addition of blastomas (Fig. 5F, a–l). In particular, the hypercellular EDN3 (1–100 nmol/L) improved the viability of GSCs as zones surrounding necrotic foci similar to histopathologic determined by the trypan blue exclusion test (50%–80% features of pseudopalisading necrosis was observed (Fig. 5F, viability; P < 0.05). However, reduced viability was observed a–d). The infiltrative tumor exhibit hypercellularity, hyper- when high doses of EDN3 (300 and 1,000 nmol/L) were chromatism, pleomorphism, mitosis, vascular endothelial added (Fig. 6A). Rescue of GSCs from apoptosis by adding hyperplasia, and oligodendroglial components were also exogenous EDN3 was further evidenced by a quantitative identified (Fig. 5F, e–l). On the other hand, injection of apoptosis assay coupled with morphologic examination (Fig. GSCs pretreated with 50 mmol/L BQ788 failed to develop 6B and C). In general, 30 nmol/L EDN3 alone lowered the a tumor (0/14) at week 25. The area of tumor injection site degree of cell apoptosis to a level close to that of SGF shows no evidence of neoplastic cells (Fig. 5F, m–r) and compared with plain media. Furthermore, low proliferative show no significant changes when compared with normal stimulation was seen with the addition of exogenous EDN3 brain tissue (Fig. 5F, s–v), except a scar-like feature is (1.5-fold; Fig. 6A), yet exogenous EDN3 alone was not occasionally observed near the injection site (Fig. 5F, r). able to propagate GSCs in culture as that of SGF media, Thesedatathussuggestthepotentialinvolvementof suggesting that the significance of "growth stimulation" (P < autocrine EDN3/EDNRB signaling in maintaining the 0.05) is likely due to significantly fewer apoptotic cells tumorigeniccapacityofGSCsinculture. maintained when compared with cells cultured in plain To investigate whether in vitro cultured GSCs change the media. This notion was further tested by investigating the þ expression of the EDN ligands and receptors, we analyzed effects of exogenous EDN3 on sorted CD133 and and compared the expression of EDN system genes in tumor CD133 daughter cells. We found that EDN3 alone could þ xenografts (initiated from purified CD133 GSCs), and enhance the viability of both types of cells in a dose–response þ CD133 and CD133 cells directly sorted from them, by fashion as determined by trypan blue staining (Fig. 6D). RT-PCR (Fig. 5G, a). CD133, EDN1, EDN3, and However, in contrast to cells cultured in SGF, sphere EDNRA mRNA were determined in tumor xenografts (Fig. formation and cell expansion were not achievable by EDN3 5G, a), whereas a lower level of EDN3 mRNA was detected alone, even when EDN3 were repetitively added daily compared with that of EDN1 mRNA. However, although (Supplementary Fig. S4). These data therefore suggest that þ EDNRB mRNA was detected in CD133 GSCs used for EDN3 alone is not a potent mitogen for GSCs but may injection, it could not be detected in tumor xenografts after provide cell survival benefits. the 30 cycles of PCR. Similar expression patterns of the EDN system genes were also shown in tumors by immu- Genes regulated by EDN3/EDNRB signaling nohistochemical staining (Fig. 5G, b). Likewise, EDN1, To gain further insight into the molecular mechanisms EDN2, EDN3, and EDNRA mRNA were also determined underlying the requirement of EDN3/EDNRB signaling in þ in both CD133 and CD133 cells sorted from tumor maintaining GSCs, we carried out a large-scale gene expres- þ xenografts, whereas CD133 cells express a lower level of sion comparison of GSCs treated with and without EDNRB EDN1 and a higher level of EDN3 mRNA when compared inhibitor (BQ788). Probe set signals on the expression array with those of CD133 cells (Fig. 5G, c). YKL-40 mRNA that were equal to less than 1.5-fold lower in 3 treated (n ¼ 3 could only be detected in tumor xenografts and sorted patients) versus 6 untreated GSC samples (50 mmol/L, 24 þ CD133 cells (Fig. 5G, a, c). When CD133 cells were hours; n ¼ 3 patients, sample duplicate) with a pairwise t test recultured in SGF media for 7 days, the expression of (P < 0.05) were selected. Samples were permuted 100 times EDNRB mRNA could be redetected (Fig. 5G, d). On the by dChip for a FDR of 11% (median), and 61 significant other hand, tumor xenografts which exhibit a higher level of genes were obtained (Table 1). The major downregulated EDN3 mRNA and lower levels of EDN1 and EDN2 genes identified among 61 genes are those involved in mRNA were also identified (Supplementary Fig. S3). Nev- organizing cytoskeleton structure and function (e.g., ertheless, a higher level of EDNRA mRNA is always EFEMP1, IQGAP1, CADL1, WASF2, S100A6, MYO10, þ expressed in tumor xenografts and sorted CD133 and MYO6, CCDC88A, RHOQ, SRGAP2, PALLD, CTSB, CD133 cells, whereas a low level of EDNRB mRNA could WASL, FRMD4B, HIP1, RAB31, and VPS35), mitotic only be detected in tumor xenografts. These data may spindle assembly, mitotic checkpoint (e.g., ASPM, suggest that the initiation of tumor growth in vivo from NUSAP1, ASPM, and USAP1), and DNA repair or anti- GSCs is accompanied with the activation/upregulation of apoptosis (e.g., UHMK1, NIPBL, KRAS, G2E3, DHX9, EDNRA signaling and downregulation of EDNRB GTSE1, RIF1, and NAA15), reflecting disruptions in cell signaling. structure, cell polarity, cell movement, intracellular

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A B E

BQ788 Posttreatment treatment reseeding Control Reagents siRNA Control EDN3 siRNA-1 EDN3 siRNA-2 No cDNA M.W. Control Reagents siRNA Control EDN3 siRNA-1 EDN3 siRNA-2 No cDNA D431 D431 S496 E445 C 400 350 Control siRNA 300 EDN3 siRNA 250

S496 200 50 μmol/L 150

EDN3 (pg/mL) 100 50 0 D D431 S496 E445 Fibroblast 0.9 0.8 Control siRNA 0.7 0.6 EDN3 siRNA-1

E445 0.5 0.4 0.3 0.2 0.1 oligonucleosomes

(absorbance 405 nm) 0 Amount of cytoplasmic D431 S496 E445 Fibroblast

ae im qs

bfjn rt

cgk o u

dh l p v

Figure 5. Blockade of EDN3/EDNRB signaling impairs GSC self-renewal, cell migration, and tumorigenic capacity. A, light-microscopic morphology of GSC cultures. The indicated GSC cultures were treated with and without BQ788 at the concentration of 50 mmol/L for 3 days and the pictures were taken (a–f). Cells were harvested and were reseed at clonal density, which prevent cells from forming aggregates. The cultures were incubated for 3 days and pictures were taken (g–l). Scale bar ¼ 25 mm. B, the indicated GSC cultures were transfected with negative control siRNA, EDN3 siRNA (clone #1 and clone#2), or treated with or without transfection reagents. Cells were incubated for 3 days and the levels of EDN3 mRNA were assayed by qRT-PCR. The GAPDH was used as an internal control gene. Fibroblasts serve as a negative control cell line, which does not express EDN3 (data not shown). C, EDN3 protein peptides concentrations secreted into the conditioned media by transfected cells were assayed by anti-EDN3 ELISA kit. Data represent mean values SD of triplicate.

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G bc a β-actin (152 bp)

CD133 (226 bp)

EDN1 (250 bp)

EDN2 (158 bp)

EDN3 (110 bp)

200 bp 100 bp EDNRA (119 bp)

EDNRB (197 bp)

YKL-40 (206 bp) M.W. β -actin (152 bp) CD133 (226 bp) EDN1 (250 bp) EDN3 (110 bp) EDNRA (119 bp) EDNRB (197 bp) YKL-40 (206 bp)

200 bp 100 bp M.W. No cDNA β -actin (152 bp) EDN1 (250 bp) EDN2 (158 bp) EDN3 (110 bp) EDNRA (119 bp) EDNRB (197 bp)

200 bp 100 bp

Figure 5. (Continued) measurements in triplicate experiments. , P < 0.05, , P < 0.001 versus control siRNA transfected cells. D, cell apoptosis in negative control siRNA or EDN3 siRNA transfected cells was determined by a cell death detection ELISA kit. Data represent mean values SD of triplicate measurements in triplicate experiments. , P < 0.05 versus control siRNA-treated cells. E, light-microscopic morphology of indicated cells treated with assay buffer (a, e, i, m), transfection reagents (b, f, j, n), negative control siRNA (c, g, k, o), and EDN3 siRNA-1 (d, h, l, p) for 72 hours, as well as negative control siRNA for 7 days (q–s). Scale bar ¼ 25 or 50 mm. F, representative photographs of hematoxylin and eosin (H&E) staining of mouse brain tissue. Brain tissues from mice injected with untreated GSCs display invasive growth of gliomas with diffuse infiltration into the surrounding tissue and vessels (a–l). Infiltrating tumor exhibits hypercellular zones surrounding necrotic foci and forms a "pseudo-palisading" necrosis pattern (S496, frozen section; a–d). Other important histopathologic features of glioblastoma are also seen in tumor lesions, including hypercellularity (e), hyperchromatism (f, g), pleomorphism and mitosis (h–j), vascular endothelial hyperplasia (k), and oligodendroglial component (l; D431, S496, E445). Mouse brains injected with GSCs pretreated with 50 mmol/L BQ788 showed no evidence of tumor development (m–r). Mouse brain injected with stem cell media serves as control for normal brain tissue (s–u). Magnification, 4 (o, s), 8 (a, m), 100 (e, f, p, t, u), 200 (b, c, g, n, q, r, v), 400 (d, h–l). G, presence of EDN system components in glioma xenografts. The expression of the indicated EDN system components in both subcutaneous and intracranial glioma xenografts were analyzed by qRT-PCR with specific primers. b-Actin was used as an internal control gene (a). Representative immunohistochemical stainings of EDN components in intracranial tumor xenografts (b). The mRNA levels of the EDN þ system components in uncultured CD133 and CD133 cells directly sorted from the glioma xenografts were analyzed by qRT-PCR (c). The expressions of EDN system transcripts in CD133þ cells recultured in SGF media for 7 days (d).

transports, cell division, and cell survival pathway. S5). Correspondingly, GSEA identified 97 significant gene Concomitantly, a series of genes that provides negative ontology (GO) clusters (Supplementary Table S1), mostly regulation of development, cell growth, and differentiation associated with microtubule cytoskeleton organization, cell were identified (e.g., IFI27, SOCS3, MTUS1, SCML1, division, motor activity, spindle, DNA packaging, leading IFI16, LUZP1, IL6ST, JMJD1C, TEAD1, LINGO1, edge, transcription repressor activity, negative regulation of C9orf86, PSD3, and USP10), suggesting the stemness prop- DNA binding, detection of chemical stimulus, cell-cycle erties and cellular quiescence in GSCs were disturbed. The arrest, and neuron development, offering a potential molec- distinctive gene expression profiles of randomly selected ular explanation for how loss of EDN3/EDNRB signaling genes were verified by qRT-PCR (Supplementary Fig. may impact GSC migration, self-renewal, cell survival, and

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EDN3 is a Survival Factor for Glioblastoma Stem Cells

ACD431 S496 E445

100 2 EDN3 EDN3 EDN3 P < 0.05 0 nmol/L 0 nmol/L 0 nmol/L 80 1.5 60 P < 0.05 1 40 1 1 1 0.5 nmol/L nmol/L nmol/L

% Viability 20 D431

0 0 Grow (fold) 3 3 3 nmol/L nmol/L nmol/L

100 P 2 < 0.05 10 10 10 nmol/L nmol/L nmol/L 80 1.5 60 P < 0.05 1 40 0.5 30 30 30 20 nmol/L nmol/L nmol/L % Viability S496

0 0 Grow (fold)

100 100 100 nmol/L nmol/L nmol/L

100 2 300 300 300 80 Xp>0.05 1.5 nmol/L nmol/L nmol/L 60 X 1 40 X X 0.5 1,000 1,000 1,000 % Viability 20 E445 nmol/L nmol/L nmol/L

0 0 (fold)Grow 0 1 3 10 30 100 300 1,000 EDN3 (nmol/L) 25 μm SGF SGF SGF

B D ** ΔΔ 100 * Δ 1.4 Δ *Δ EDN3 (nmol/L) 0 1 30 100 1,000 SGF 80 1.2 * 1 * 60 0.8 + +

+ iability

V 40 0.6 CD133+ D431 % 0.4 20 CD133 – D431

oligonucleosoes 0.2 (absorbancenm) 40

Amountcytoplasmic of 0 0 D431 S496 E445 300 100 1,000 SGF EDN3 (nmol/L)

Figure 6. EDN3 is a survival factor for GSCs. A, the indicated GSC lines were cultured in the plain media supplemented with various doses of EDN3 as indicated. Cells were incubated for 3 days and viability was determined by manually counting the trypan blue-stained samples in a hemacytometer chamber and present as percentage of viable cells in cultures (D). The cell growth was determined by MTS/PMS cell proliferation assays and present as fold increase compared with nontreated cells (&). Data represent mean values SD of triplicate measurements in triplicate experiments. XP > 0.05 versus cells cultured in plain media. B, cell apoptosis in (A) was assayed by a cell death detection ELISA kit. Data represent mean values SD of triplicate measurements in triplicate experiments. , P < 0.05 versus D431 and S496 GSCs treated with 1, 30, 100, 1000 nmol/L EDN3 and SGF media. ~, P < 0.05 versus E445 cells treated with 1, 30, 100 nmol/L EDN3 and SGF media. þ, P < 0.05 versus D431, S496, and E445 cultured in SGF media. C, light-microscopic morphology of cell cultures in (A). Scale þ bar ¼ 25 mm. D, cell survival was assayed in sorted CD133 and CD133 GSC cells cultured in the SGF, plain media, or plain media supplemented with various doses of EDN3 as indicated. Cells were incubated for 7 days and viability was determined by trypan blue staining. Data represent mean values SD of triplicate measurements in triplicate experiments. , P < 0.05, , P < 0.01 (CD133þ cells), D, P < 0.05, DD, P < 0.01 (CD133 cells) versus cells cultured in plain media.

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tumorigenic capacity. We used the default parameters of in response to differentiation, indicating that EDN3 and GSEA and Gene Ontology gene sets were permuted 1,000 EDN1 genes can be epigenetically regulated, and that times. We called a gene set significant if the FDR Q value was increased EDN1 may be associated with the onset of under 0.15. BQ788 works by potently and competitively tumorigenesis. Indeed, we detected an increased expression inhibiting EDN3 binding to EDNRB; therefore, it may of EDN1, but not EDN3, mRNA in patient glioblastoma explain that downregulation of EDNRB transcripts by tumors (compared with GSCs), suggesting that EDN3 may BQ788 treatment was not determined either by expression play a tumor suppressor-like role to maintain the quies- microarray or RT-PCR (data not shown). However, several cence and tumorigenic potential of GSCs. In agreement GSC genes (e.g., EFEMP, IQGAP1, SOCS3, WAVE2, with this, a frequent loss of EDN3 expression in breast MYO10, and K-RAS) downregulated by BQ788 treatment tumors due to epigenetic inactivation has previously been have been previously reported to be linked to activation of suggested (29). Likewise, there were decreased levels of mitogen-activated protein kinase (MAPK) pathways (Sup- EDN3 and increased levels of EDN1 and EDN2 mRNA in plementary Table S2), which are the downstream effector cancerous cervical epithelial cells compared with normal pathways of EDNRB signaling pathway (38–43), suggesting cervical epithelial cells (28). Thus, the reduction of EDN3 that the identified genes characterizing stemness, migration, levels (e.g., induced by exogenous cues) may implicate the and survival of GSCs are regulated by EDN3/EDNRB initiation of proliferative differentiation from quiescent signaling. GSCs toward more differentiated progeny, by which the proliferative progeny gradually overpopulate a tumor. It Discussion seemsthatsoleEDN3/EDNRBsignalingalonecannot grant tumorigenic potential to GSCs, yet the removal of The finding of EDN3 overexpression in GSCs through EDN3/EDNRB signaling contributed undifferentiated, the expression microarray analysis led to our novel investi- antiapoptotic, and migratory properties to GSCs, which gation of the EDN system in cultured GSCs. In normal may weaken or eliminate their tumorigenic capacity. Auto- þ NCC cultures, EDN3/EDNRB signaling prevents the pre- crine EDN3 in EDNRB GSCs, may therefore, play a role mature differentiation of crest-derived precursors (22, 44). in antidevelopmental, antidifferentiating, antiapoptotic, In vivo, EDN3/EDNRB signaling is required for NCC and antitumorigenic functions, thereby help in maintain- migration during ENS development (22). Mutations in ing a continuous GSCs pool. EDN3 or EDNRB can lead to the abnormal development WefoundthatlossofEDN3productioninGSCs of ENS and melanocytes, implying that the loss of function through serum-induced differentiation did not cause cell in these 2 genes may interfere with tumor development by apoptosis, whereas directly blocking EDN3/EDNRB sig- EDN3/EDNRB signaling-dependent GSCs. Indeed, block- naling causes severe GSC apoptosis. These observations ade of EDN3/EDNRB signaling of GSCs leads to the loss of seem to support the notion that autocrine EDN3/EDNRB tumorigenic potential in our GSC model. We found that signaling only provides survival benefits when GSCs are cultured GSCs express both neural and mesenchymal sig- maintained in SGF condition. A previous study has shown natures characterizing NCC-like cells and, therefore, that stimulation of the EDNRB in astrocyte induces cAMP hypothesized that autocrine EDN3/EDNRB signaling may response element-binding protein (CREB) and c-fos be a survival/maintenance factor for GSCs. In fact, when expression via multiple MAPK signaling pathways, includ- GSCs undergo serum-induced proliferative differentiation, a ing the extracellular regulated kinase (ERK) 2, c-Jun N- significant loss of EDN3 production accompanied by terminal kinase (JNK1), and p38 kinase (38). EDN3 codownregulation of a series of transcripts related to NCCs, stimulation also rapidly increased phosphorylation of RGCs, and NSCs was observed. For instance, SOX2 is a ERK2 in neural progenitor cells (39) and activated IkappaB marker of NSCs and is essential for maintaining the plu- and MAPK in the human colonic epithelial cells (40). ripotent, self-renewal, and undifferentiated phenotypes of Likewise, EDN3 treatment resulted in the activation of embryonic stem cells. Moreover, silencing of SOX2 in GSCs MAPK-p90 ribosomal S6 kinase-CREB and cAMP-pro- can cause the loss of tumorigenicity (45). FABP7, a glial- tein kinase A-CREB pathways in melanocyte culture (41, specific marker, is a direct target of Notch signaling in RGCs, 42) and induction of ERK1/2 and focal adhesion kinase and the involvement of Notch signaling in the maintenance phosphorylation in melanoma cells (43). One important of the tumorigenic potential of GSCs has been reported (46). note is that the activation of EDNRB by EDN3 also leads Thus, autocrine EDN3/EDNRB signaling may prevent to loss of expression of the cell adhesion molecule E- GSCs from differentiating prematurely. cadherin and associated catenin proteins, while increasing Direct evidence of the differentiation-inhibiting effect of Snail and N-cadherin expression (43). Thus, the expression EDN3 was shown in clonogenic cultures of ENS progeni- profiles of BQ788-treated GSCs supports the notion that tors supplemented with EDN3, by which neuronal and these pathways are the downstream effector pathways of glial differentiation pathways were blocked and the multi- EDN3/EDNRB signaling pathway that contribute GSC potent state of progenitors was maintained (44, 47). These survival and migration (Supplementary Table S2). The observations thus support the view that autocrine EDN3 usage of an inducible knockdown of EDNRB would maintains the undifferentiated state of GSCs. Moreover, strengthen the role of EDNRB signaling in GSCs. More- EDN3 and EDN1 mRNAs are altered reciprocally in GSCs over, because ex vivo treatment with BQ788 may already

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Table 1. Genes expressed at lower levels in BQ788-treated GSCs compared with untreated GSC

Gene Gene Symbol Fold P Functional involvement ID change

IFNa-inducible protein 27 3429 IFI27 4.30 0.029619 Antigrowth, antidifferentiation, apoptosis Heparan sulfate (glucosamine) 9957 HS3ST1 4.00 0.003801 Synthesis of anticoagulant heparin, 3-O-sulfotransferase 1 antitumor EGF-containing ﬁbulin-like 2202 EFEMP1 2.89 0.040817 Cell adhesion and migration extracellular matrix protein 1 Asp (abnormal spindle)-like, 259266 ASPM 2.61 0.004877 Mitotic spindle regulation microcephaly associated IQ motif containing GTPase 8826 IQGAP1 2.45 0.000015 Modulation of cell architecture activating protein 1 Suppressor of cytokine signaling 3 9021 SOCS3 2.44 0.000182 Negative regulators of cytokine signaling, tumor suppressor Caldesmon 1 800 CALD1 2.41 0.038236 Smooth muscle and nonmuscle contraction Mitochondrial tumor suppressor 1 57509 MTUS1 2.36 0.002229 Tumor suppressor and antimitosis Sex comb on midleg-like 1 6322 SCML1 2.28 0.000178 Control of embryonic development (Drosophila) Mex-3 homolog A 92312 MEX3A 2.26 0.002964 Involvement in posttranscriptional regulatory mechanisms IFNg-inducible protein 16 3428 IFI16 2.25 0.008661 Antigrowth, cellular quiescence, apoptosis WAS protein family, member 2 10163 WASF2 2.22 0.005534 Cell shape and motility Topoisomerase (DNA) I 7150 TOP1 2.20 0.002516 Altering the topologic states of DNA during transcription U2AF homology motif 127933 UHMK1 2.20 0.002059 Prevent senescence in G0/G1; (UHM) kinase 1 cell migration S100 calcium binding 6277 S100A6 2.19 0.032921 Cell migration/motility, antisenescence protein A6 (calcyclin) Nipped-B homolog (Drosophila) 25836 NIPBL 2.17 0.000182 Tissue development, sister chromatid cohesion, DNA repair Synaptotagmin I 6857 SYT1 2.16 0.026320 Vesicular trafﬁcking and exocytosis Myosin X 4651 MYO10 2.16 0.012113 Filopodia formation Myosin VI 4646 MYO6 2.15 0.002580 Intracellular vesicle and organelle transport Low density lipoprotein receptor 3949 LDLR 2.14 0.012743 Cholesterol synthesis CCDC88A coiled-coil domain 55704 CCDC88A 2.12 0.006578 Actin organization, cell motility/migration containing 88A Leucine zipper protein 1 7798 LUZP1 2.12 0.015862 Embryonic development of brain v-Ki-ras2 Kirsten rat sarcoma 3845 KRAS 2.10 0.001864 Possessing intrinsic GTPase viral oncogene homolog activity, antiapoptosis Ectodermal-neural cortex 8507 ENC1 2.06 0.020856 Actin-binding protein; oxidative (with BTB-like domain) stress response RNA binding motif protein 8A 9939 RBM8A 2.06 0.004386 Coupling pre- and post-mRNA splicing events DEP domain containing 1 55635 DEPDC1 2.05 0.007013 Cancer/testis antigen

G2E3 G2/M-phase–speciﬁc 55632 G2E3 2.04 0.004153 Prevention of apoptosis in E3 ubiquitin protein ligase early embryogenesis ADP-ribosylation factor-like 7 10123 ARL4C 2.02 0.022123 Cholesterol transport Secretory carrier membrane 9522 SCAMP1 2.02 0.011002 Carriers to cell surface protein 1 in post-golgi recycling pathways Formin-like 2 114793 FMNL2 1.99 0.002077 Morphogenesis, cytokinesis, and cell polarity Ras homolog gene family, 23433 RHOQ 1.96 0.005201 Myoﬁbril assembly, exocytic vesicle member Q fusion, glucose uptake (Continued on the following page)

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Table 1. Genes expressed at lower levels in BQ788-treated GSCs compared with untreated GSC (Cont'd )

Gene Gene Symbol Fold P Functional involvement ID change

Interleukin 6 signal 3572 IL6ST 1.94 0.011686 Embryonic development transducer (gp130) DEAH (Asp-Glu-Ala-His) 1660 DHX9 1.93 0.006616 DNA repair, genome stability box polypeptide 9 Oxysterol binding protein-like 8 114882 OSBPL8 1.93 0.001205 Intracellular lipid receptors THO complex 2 57187 THOC2 1.92 0.000834 Binds to spliced mRNAs to facilitate mRNA export Zinc finger protein 533 151126 ZNF385B 1.92 0.009225 Neuronal function; RNA maturation and stability G-2 and S-phase expressed 1 51512 GTSE1 1.91 0.007537 Antiapoptosis, control DNA damage Nucleolar and spindle 51203 NUSAP1 1.90 0.001876 Spindle microtubule organization associated protein 1 Jumonji domain containing 1C 221037 JMJD1C 1.90 0.001287 Reactivation of silenced genes in undifferentiated ES cells TEA domain family member 1 7003 TEAD1 1.88 0.001749 Transcriptional repressor; antidifferentiation SLIT-ROBO Rho GTPase 23380 SRGAP2 1.87 0.000640 Controlling cell spreading activating protein 2 and cell migration Adaptor-related protein complex 1, 8905 AP1S2 1.86 0.009349 Recognition of sorting signals sigma 2 subunit Palladin 23022 PALLD 1.85 0.003542 Control of cell shape, adhesion, and contraction Leucine rich repeat neuronal 6A 84894 LINGO1 1.85 0.002910 Negative regulator of oligodentrocyte differentiation Cathepsin B 1508 CTSB 1.82 0.007249 Invasion, migration Wiskott–Aldrich syndrome-like 8976 WASL 1.81 0.009275 Induces actin polymerization and redistribution Chromosome 9 open reading 55684 C9orf86 1.81 0.001019 Cell growth regulation frame 86 HIV TAT–specific factor 1 27336 HTATSF1 1.79 0.007012 Process of transcriptional elongation Pleckstrin and Sec7 23362 PSD3 1.79 0.003251 Tumor suppressor domain containing 3 FERM domain containing 4B 23150 FRMD4B 1.79 0.004509 Cytoskeletal signals and membrane dynamics RAP1 interacting factor 55183 RIF1 1.76 0.006310 DNA repair homolog (yeast) Discoidin, CUB and LCCL 285761 DCBLD1 1.76 0.011326 Integral to membrane, cell adhesion domain containing 1 Glutamyl-prolyl-tRNA synthetase 2058 EPRS 1.75 0.000371 Catalyzes the aminoacylation Male sterility domain containing 2 84188 FAR1 1.74 0.005190 Controls ether glycerophospholipid synthesis Huntingtin interacting protein 1 3092 HIP1 1.70 0.002967 Membrane-cytoskeletal integrity NMDA receptor regulated 1 80155 NAA15 1.68 0.003107 N-acetyltransferase, antiapoptosis Ubiquitin-specific peptidase 10 9100 USP10 1.62 0.001657 Cleaves ubiquitin, a regulator of p53, antitumor Coiled-coil domain containing 75 253635 CCDC75 1.62 0.000894 Unknown RAB31, member RAS 11031 RAB31 1.62 0.001205 Vesicle and granule targeting oncogene family Hepatitis B virus x associated protein 51773 RSF1 1.57 0.002448 Chromatin remodeling Vacuolar protein sorting 35 (yeast) 55737 VPS35 1.54 0.000354 Subunit of the retromer, vesicle transport

NOTE: Probe set signals on the expression array that were <1.5-fold lower in BQ788 treated (n ¼ 3 patients) versus untreated GSC samples (n ¼ 3 patients, sample duplicate) with a pairwise t test (P < 0.05) were selected. Samples were permutated 100 times by dChip and 61 genes with median FDR ¼ 11% were obtained.

1682 Mol Cancer Res; 9(12) December 2011 Molecular Cancer Research

EDN3 is a Survival Factor for Glioblastoma Stem Cells

damage the capabilities of self-renewal and proliferative same sphere cultures contain enriched tumorigenic cells, yet differentiation in GSCs, inducible knockdown of EDNRB they express tumor suppressor phenotype (8). Thus, true or EDN3 using viral-delivered hairpin RNA would allow GSCs may be quiescent before undergoing asymmetric cell more in depth functional and in vivo studies. division to simultaneously self-renew and generate more It has been shown that EDN3 first stimulates expression differentiated progeny. It seems that differentiated progeny of EDNRB, then when under prolonged exposure to EDN3, are active-growing cells that make up the most population in EDNRB expression decreases (12). This may explain why tumor spheres and are likely the effector progeny which can EDNRB are expressed at lower levels in the sphere cultures, undergo proliferative differentiation to produce more dif- which consistently produce EDN3. We found that EDN3 ferentiated progeny that expresses hyperproliferative and alone is not a potent mitogen for GSCs and similar finding hyperangiogenic phenotype, leading to tumor formation in was also reported (44). Interestingly, it was shown that animals. Therefore, identifying essential genes (e.g., EDN3) EDNRB antagonists reduced the viability and proliferation which are shared by both GSCs and their immediate of glioma cells, which do not express EDN3 (48), implying differentiated progeny/daughter cells would enhance its the possibility of blocking EDN1/EDNRB signaling. The clinical value because both populations can be targeted. decreased glioma cell viability by EDNRB antagonists inde- Thus, treatment with BQ788 not only abolishes the self- pendent of their cognate receptor was also reported (49). We renewal capacity of quiescent GSCs but also prevents dif- speculate that low levels of EDNRB mRNA being expressed ferentiated progeny from populating tumor spheres and þ in cultured CD133 GSCs used for injection, but not in forming tumor in animals. þ some tumor xenografts, could be due to tumor initiation Depletion of EDN3 cells will likely prevent either þ þ from CD133 GSCs being accompanied by activation/ CD133 or CD133 GSCs from regenerating a tumor. upregulation of EDNRA signaling and downregulation of Futures studies should determine whether addition of EDNRB signaling in our model system. Alternatively, it is EDNRB antagonist can sensitize cells to radiochemotherapy possible that EDNRB may be only expressed by a small in treatment of established glioblastoma tumors in animals. subset of quiescent GSCs that express the tumor-suppressive Our data support the view that the prevention of GSC- þ þ phenotype (e.g., CD133 /EDNRB cells) for maintaining mediated tumor recurrence may need to focus on targeting GSCs pool in vivo, which can be retrieved and enriched by active stem cell pathways in GSCs (such as EDN3/EDNRB þ SGF culturing. On the other hand, CD133 cells sorted pathway), not proliferative pathways. Obviously, the cure for from xenografts may mostly contain activated GSCs (e.g., brain cancer requires eliminating both GSC and non-GSC þ þ CD133 /EDNRB /EDNRA cells) that have entered the populations; therefore, it is important to evaluate the syn- pathway to tumorigenesis. Interestingly, some tumor xeno- ergistic benefits of incorporating GSC-targeted therapies grafts express high levels of EDN3 and EDNRA mRNA, but into conventional cancer treatments. low levels of EDN1 and EDN2 mRNA, with a negligible level of EDNRB mRNA, suggesting that the growth of fl þ Disclosure of Potential Con icts of Interest EDN3 tumor may not be EDNRB, but EDNRA signaling dependent. Promoter hypermethylation of the EDNRB gene No potential conflicts of interest were disclosed. in various human malignancies has been reported and suggested that EDNRB may be a candidate tumor suppres- Grant Support sor gene (50). Finally, our genome-wide expression analysis has provided This work was supported by grants from the American Cancer Society (RSG- 07-109-01-CCE), National Cancer Institute (1 R21 CA140912-01), NIH some molecular explanation for the requirement of EDN3/ (1DP2OD006444-01), and The Bradley Zankel Foundation to C-L Tso. EDNRB signaling in maintaining GSC sphere cultures. The costs of publication of this article were defrayed in part by the payment of page Apparently, EDN3/EDNRB blockade mostly impacts cell charges. This article must therefore be hereby marked advertisement in accordance with structure, cell movement, self-renewal/cell division, and cell 18 U.S.C. Section 1734 solely to indicate this fact. survival, rendering GSCs nontumorigenic. We previously þ Received December 12, 2010; revised September 21, 2011; accepted October 2, showed that CD133 cells, not CD133 cells, sorted from 2011; published OnlineFirst October 19, 2011.

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Autocrine Endothelin-3/Endothelin Receptor B Signaling Maintains Cellular and Molecular Properties of Glioblastoma Stem Cells

Yue Liu, Fei Ye, Kazunari Yamada, et al.

Mol Cancer Res 2011;9:1668-1685. Published OnlineFirst October 19, 2011.

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