Gene Alterations Identified by Expression Profiling in Tumor-Associated Endothelial Cells from Invasive Ovarian Carcinoma

Research Article

Chunhua Lu,1 Tomas Bonome,3 Yang Li,1 Aparna A. Kamat,1 Liz Y. Han,1 Rosemarie Schmandt,1 Robert L. Coleman,1 David M. Gershenson,1 Robert B. Jaffe,4 MichaelJ. Birrer, 3 and AnilK. Sood 1,2

Departments of 1Gynecologic Oncology and 2Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; 3Cell and Cancer Biology Branch, National Cancer Institute, Bethesda, Maryland; and 4Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California

Abstract the promise of such approaches. However, the full spectrum of Therapeutic strategies based on antiangiogenic approaches differences in the tumor vasculature compared with its normal are beginning to show great promise in clinical studies. counterpart is not known. Identification of additional targets on However, full realization of these approaches requires tumor endothelium may allow opportunities for developing new identification of key differences in gene expression between therapeutic approaches to inhibit angiogenesis in a tumor-specific endothelial cells from tumors versus their normal counter- manner. parts. Here, we examined gene expression differences in Higher levels of proangiogenic cytokines and angiogenesis are purified endothelial cells from 10invasive epithelial ovarian associated with an increased risk of metastasis and poor prognosis cancers and 5 normal ovaries using Affymetrix U133 Plus in ovarian cancer (5, 6). To date, a small number of breast, colon, 2.0microarrays. More than 400differentially expressed genes and brain cancers have been analyzed for gene expression changes were identified in tumor-associated endothelial cells. We in the tumor vasculature using serial analysis of gene expres- selected and validated 23 genes that were overexpressed by sion (7–9). These studies showed the ability to define both tumor- 3.6- to 168-fold using real-time reverse transcription-PCR specific endothelial genes and normal endothelial genes. Whereas and/or immunohistochemistry. Among these, the polycomb selected genes in ovarian cancer vasculature have been character- group protein enhancer of Zeste homologue 2 (EZH2), the ized, there is little information about global gene expression Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold alterations in ovarian cancer endothelium. This lack of data in tumor-associated endothelial cells. Silencing these genes prompted us to carry out expression profiling on purified endothelial individually with small interfering RNA blocked endothelial cells from invasive epithelial ovarian cancers and normal ovaries. cell migration and tube formation in vitro. The present study In recent years, whole genome expression profiling of cancer shows that tumor and normal endothelium differ at the using methods such as microarray and serial analysis of gene molecular level, which may have significant implications for expression has advanced our understanding of the molecular the development of antiangiogenic therapies. [Cancer Res pathways involved in cancer onset and progression. However, 2007;67(4):1757–68] global analysis of gene expression in specific cell populations within the tumor microenvironment is challenging and bulk tissue Introduction expression profiling may, in fact, mask gene changes in different cell types. We have recently used laser capture microdissection to Despite improvements in surgery and chemotherapy, mortality isolate epithelial cells from ovarian cancers for microarray analyses rates in women with advanced ovarian carcinoma have remained (10), which elucidated changes in gene expression specific to the largely unchanged (1). Therefore, novel therapeutic strategies are epithelial tumor cells. Profiling expression changes that occur in needed. It is now well known that growth of tumors, both at the the tumor stroma, including the tumor endothelial cells, will likely primary and metastatic sites, requires a blood supply for expan- provide insights into the mechanisms underlying tumor vascular sion beyond 1 to 2 mm (2). Targeting tumor angiogenesis by inhi- growth, reveal additional targets for antiangiogenic therapies, and biting endothelial cells that support tumor growth is particularly potentially offer new biomarkers for diagnosis and surveillance. promising because of their presumed genetic stability. The recent However, the endothelium is enmeshed in a tissue complex success of a humanized monoclonal antibody (bevacizumab) consisting of vessel wall components, stromal cells, and epithelial against vascular endothelial growth factor in prolonging the lives cells. Only a small fraction of the cells within these tissues are of patients with advanced colon and breast carcinoma (3, 4) shows endothelial. Moreover, gene analysis of specific cell types extracted from chemical reagent–fixed frozen tissue may not be accurate as the gene profile may be altered during the fixation process. In the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). present study, we immunopurified endothelial cells from human C. Lu and T. Bonome contributed equally to this work. M.J. Birrer and A.K. Sood normal ovarian tissues and invasive epithelial cancers and share senior authorship. investigated the gene expression profile using microarrays. Selected Requests for reprints: Anil K. Sood, Department of Gynecologic Oncology, University of Texas M. D. Anderson Cancer Center, Unit 1362, 1155 Herman Pressler, genes were validated to test the reliability of the microarray Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood@ analysis. The gene expression profiles derived in the current study mdanderson.org. I2007 American Association for Cancer Research. define unique alterations in vascular gene expression in epithelial doi:10.1158/0008-5472.CAN-06-3700 ovarian carcinoma. www.aacrjournals.org 1757 Cancer Res 2007; 67: (4).February 15, 2007

Materials and Methods as the actual data set at P < 0.001 was reported as the significance level of the global test. Sample preparation. Fresh tissue samples (5 normal ovaries and 10 Pathway analysis. Differentially regulated genes identified in a series of epithelial high-grade, stage III or IV invasive serous ovarian cancers) were 48 late-stage (III and IV), high-grade (3) microdissected papillary serous obtained from patients undergoing primary surgical exploration at the ovarian carcinomas, as compared with 10 normal ovarian surface epithelial M. D. Anderson Cancer Center after approval from the Institutional Review brushings (10), were categorized by cellular component according to the Board. The minced tissue was digested with collagenase A, elastase, and Gene Ontology ontological hierarchy. Epithelial genes associated with the j DNase 1 at 37 C for 90 min to yield a single-cell suspension. A number of cell membrane, extracellular matrix, and extracellular region were used as negative selections followed, including removal of platelets and RBCs by central nodes to identify signaling pathways modulated in tumor-associated Percoll separation; removal of epithelial cells using M450 beads, which are endothelial cell isolates. This was accomplished using PathwayAssist prebound to BerEP4 antibody; and removal of leukocytes using anti-CD14, version 3.0 software (Iobion Informatics LLC, La Jolla, CA). This software anti-CD45, and anti-CD64 beads (Dynal Biotech, Brown Deer, WI). Positive package contains more than 500,000 documented protein interactions selection was done with P1H12 (CD146) immunobeads (P1H12 antibody acquired from MedLine using the natural language processing algorithm was from Chemicon, Temecula, CA), and the beads linked to secondary MEDSCAN. The proprietary database can be used to develop a biological antibody were from Dynal Biotech. Immunostaining was then done using association network to identify putative coregulated signaling pathways von Willebrand factor and 4¶,6-diamidino-2-phenylindole nuclear staining to using expression data. confirm the purification of endothelial cells. Quantitative real-time PCR validation. Quantitative real-time RT-PCR Total RNA amplification for Affymetrix GeneChip hybridization and was done on 100 ng of double-amplified product from the 15 specimens image acquisition. To successfully generate sufficient labeled cRNA for using primer sets specific for 23 select genes and the housekeeping genes microarray analysis from 25 ng of total RNA, two rounds of amplification GAPDH, GUSB, and cyclophilin. An iCycler iQ Real-time PCR Detection were necessary. For the first-round synthesis of double-stranded cDNA, System (Bio-Rad Laboratories, Hercules, CA) was used in conjunction with 25 ng of total RNA were reverse transcribed using the Two-Cycle cDNA the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Inc., Valencia, CA) ¶ Synthesis Kit (Affymetrix, Santa Clara, CA) and oligo-dT24-T7 (5 -GGCC- according to previously described cycling conditions (13). To calculate the AGTGAATTGTAATACGACTCACTATAGGGAGGCGG-3¶) primer according ÀDDC relative expression for each gene, the 2 T method was used, averaging to the manufacturer’s instructions followed by amplification with the MEGA the CT values for the three housekeeping genes for a single reference gene script T7 Kit (Ambion, Inc., Austin, TX). After cleanup of the cRNA with a value (14). GeneChip Sample Cleanup Module IVT column (Affymetrix), second-round Immunohistochemical staining. Paraffin sections were stained for the double-stranded cDNA was amplified using the IVT Labeling Kit following antibodies: rabbit anti-Fyn at 1:400 (Santa Cruz Biotechnology, (Affymetrix). A 15.0-Ag aliquot of labeled product was fragmented by heat Inc., Santa Cruz, CA), rabbit anti–focal adhesion kinase (FAK) at 1:50, mouse and ion-mediated hydrolysis at 94jC for 35 min in 24 ALofHO and 6 ALof 2 anti–matrix metalloproteinase (MMP)-9 at 1:40 (Oncogene Research 5Â Fragmentation Buffer (Affymetrix). The fragmented cRNA was Products, Boston, MA), anti–h2-arrestin at 1:200 (Santa Cruz Biotechnol- hybridized for 16 h at 45jC in a Hybridization Oven 640 to a U133 plus ogy), anti-PLXDC1 at 1:200 (Abcam, Inc., Cambridge, MA), or anti-Jagged1 at 2.0 oligonucleotide array (Affymetrix). Washing and staining of the arrays 1:200 (Santa Cruz Biotechnology) diluted in PBS at 4jC. After three washes with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR) in PBS, sections were incubated with secondary antibody for 1 h at room was completed in a Fluidics Station 450 (Affymetrix). The arrays were then temperature. Positive reactions were rendered visible by incubating the scanned using a confocal laser GeneChip Scanner 3000 and GeneChip slides with stable 3,3-diaminobenzidine for 5 to 10 min. Sections were Operating Software (Affymetrix). rinsed with distilled water, counterstained with Gill’s hematoxylin for 30 s, Data normalization and filtering. Global normalization at a target and mounted with Universal Mount (Research Genetics, Huntsville, AL). value of 500 was applied to all 15 arrays under consideration using The intensity of protein expression in the endothelial cells was evaluated GeneChip Operating Software (Affymetrix). Normalized data were uploaded using OPTIMAS 6.5 software and the mean absorbance was calculated from into the National Cancer Institute Microarray Analysis Database for quality five normal ovarian tissue and five ovarian cancer samples. Ten vessels were control screening and collation before downstream analyses.5 Biometric randomly selected from each sample for the measurements. Research Branch (BRB) ArrayTools version 3.2.2 software, developed by Small interfering RNA. The small interfering RNA (siRNA) constructs Drs. Richard Simon and Amy Peng Lam of the Biometrics Research Branch were purchased from Qiagen (Germantown, MD): a control sequence with of the National Cancer Institute, was used to filter and complete the statis- no homology to any human mRNA (as determined by BLAST search) and tical analysis of the array data. BRB-ArrayTools is a multifunctional Excel separate sequences designed to target EZH2, Jagged1,orPTK2 mRNA. add-in that contains utilities for processing and analyzing microarray data The EZH2 siRNA was targeted to the region corresponding to residues using the R version 2.0.1 environment (R Development Core Team, 2004). Of 85 to 106 of human EZH2 (NM004456). The Jagged1 siRNA target sequence the 47,000 transcripts represented on the array, hybridization control probe is 5¶-CTGCATTTAGGGAGTATTCTA-3¶.Forin vitro delivery, siRNA (5 Ag) sets and probe sets scored as absent at a = 0.05 or marginal at a = 0.065 1 2 was incubated with 30 AL of RNAiFect transfection reagent (Qiagen) for were excluded. In addition, only those transcripts present in >50% of the 10 min at room temperature and added to cells in culture at 80% con- arrays and displaying a variance in the top 50th percentile were evaluated. fluence in 35-mm culture plates. Class comparison analysis. Differentially expressed genes were Cell migration assay. Unstimulated motility was determined in identified for tumor and normal endothelial cell specimens using a membrane invasion culture system chambers containing polycarbonate multivariate permutation test in BRB-ArrayTools (11). A total of 2,000 filter (with 10-Am pores) that had been soaked in 0.1% gelatin, as previously permutations were completed to identify the list of probe sets with a false described (15). Human umbilical vein endothelial cells (HUVEC; 1 Â 105) discovery rate of <10% at a confidence of 95%. Differential expression was were seeded in each upper well, allowed to incubate at 37jC for 6 h in considered significant at P < 0.001. A random-variance t test was selected to DMEM containing 15% serum, and subsequently processed as described for permit the sharing of information among probe sets within class variation the invasion assay. without assuming that all of the probe sets possess the same variance (12). Tube formation assay. Matrigel (12.5 mg/mL) was thawed at 4jC and A global assessment of whether expression profiles were different between 50 AL were quickly added to each well of a 96-well plate and allowed to classes was also done. During each permutation, the class labels were solidify for 10 min at 37jC. The wells were then incubated for 6 h at 37jC reassigned randomly and the P value for each probe set was recalculated. with HUVECs (20,000 per well), which had previously been treated for The proportion of permutations yielding at least as many significant genes 18 h with the indicated siRNA. The formation of capillary-like structures was examined microscopically and photographs (Â50) were taken using a Retiga 1300 camera and a Zeiss Axiovert S100 microscope. The extent to 5 http://nciarray.nci.nih.gov/index.shtml which capillary-like structures formed in the gel was quantified by analysis

Cancer Res 2007; 67: (4).February 15, 2007 1758 www.aacrjournals.org

Figure 1. A, immunofluorescence (von Willebrand factor and Hoechst) staining of endothelial cells isolated from human normal ovary and ovarian cancer tissues. Endothelial cells were isolated from human normal ovarian and invasive epithelial ovarian cancer tissues by negative and positive immunoselection procedures as described in Materials and Methods. All photomicrographs were taken at original magnification (Â100). B, quantitative real-time PCR validation of endothelial cell microarray data. Gene expression in 10tumor isolates was calculated as mean fold change relative to five normal endothelial specimens (normal = 1) us ing the ÀDD 2 CT method. Real-time validation confirmed significant differential expression of 23 up-regulated genes, including HES4, FYN, VAV2, PGF, ECGF1, PTK2, TNFAIP6, EZH2, EGFL6, STC1, CSPG2, ADAM12, COL5A3, COL18A1, PCOLCE, PMAIP1, CENTA2, MMP9, NPTX2, TMEPAI, ARRB2, JAG1, and PLXDC1, in tumor endothelial cells versus normal isolates. Of these genes, many (e.g., VAV2, TNFAIP6,andEZH2) have not been previously described in endothelial cells. C, immunohistochemical staining for PTK2, Fyn, MMP9, h2-arrestin, Jagged1, and PLXDC1 in human normal ovarian and invasive epithelial ovarian cancers. All photomicrographs were taken at original magnification (Â100 or Â200). D, the intensity of protein expression in the endothelial cells from five normal ovarian and five ovarian cancer samples was evaluated using the OPTIMAS 6.5 software. For each protein, the intensity of staining in the normal ovarian samples was set at 1 and the fold change was calculated for the tumor endothelium. *, P < 0.01; **, P < 0.001. www.aacrjournals.org 1759 Cancer Res 2007; 67: (4).February 15, 2007

Table 1. Genes up-regulated by z6-fold in the tumor-associated endothelium

Entrez gene ID Gene Description Fold Chromosomal Function difference location

25975 EGFL6 EGF-like domain, multiple 6 36.8 Xp22 May regulate cell cycle and oncogenesis 7130 TNFAIP6 Tumor necrosis factor, a-induced 29.1 2q23.3 Anti-inflammatory and protein 6 chondroprotective effect 7291 TWIST1 Basic helix-loop-helix 19.0 7p21.2 Inhibits chondrogenesis transcription factor 6781 STC1 Stanniocalcin 1 13.3 8p21–p11.2 Regulates calcium/phosphate homeostasis and cell metabolism 84525 HOP Homeodomain-only protein, 13.1 4q11–q12 Transcriptional repressor; transcript variant 2 modulates serum response factor–dependent cardiac- specific gene expression and cardiac development 1462 CSPG2 Chondroitin sulfate proteoglycan 10.4 5q14.3 Extracellular matrix component 2 (versican) of the vitreous gel; anti–cell adhesive 57125 PLXDC1 Plexin domain containing 1 10.2 17q21.1 Unknown 6696 SPP1 Secreted phosphoprotein 1 9.5 4q21–q25 Expressed during embryogenesis, (osteopontin, bone sialoprotein I, wound healing, and early T-lymphocyte activation 1) tumorigenesis; regulates the assembly of heterotypic fibers composed of both type 1 and type V collagen 4318 MMP9 Matrix metallopeptidase 9 9.4 20q11.2–q13.1 Breakdown of extracellular (gelatinase B, 92-kDa type IV matrix; plays a role in collagenase) angiogenesis and tumor invasion 3937 LCP2/SLP76 Lymphocyte cytosolic protein 2 8.7 5q33.1–qter Promotes T-cell development (SH2 domain containing leukocyte and activation protein of 76 kDa) 152189 CKLFSF8 Chemokine-like factor superfamily 8 8.6 3p23 Regulates EGF-induced signaling; regulates cell proliferation 5366 PMAIP1 Phorbol-12-myristate-13-acetate– 8.5 18q21.32 Unknown induced protein 1 24147 FJX1 Four jointed box 1 (Drosophila) 7.7 11p13 In Drosophila, a downstream target of the Notch signaling pathway, regulates cell growth and differentiation; not known in human 8038 ADAM12 ADAM metallopeptidase domain 7.6 10q26.3 Critical for tumor development; 12 (meltrin a) involved in cell-cell and cell-matrix interactions 9636 GIP2 IFNa-inducible protein 6.9 1p36.33 Unclear; may be related to (clone IFI-15K) regulation of cell proliferation and differentiation 25878 MXRA5 Matrix-remodeling associated 5 6.9 Xp22.33 Matrix remodeling 1123 CNH1 Chimerin (chimaerin) 1 6.9 2q31–q32.1 Rho GTPase activating protein 3310 HSPA6 Heat shock 70-kDa protein 6 6.8 1q23 Involved in protein conformational (HSP70B) interactions 11211 FZD10 Frizzled homologue 10 (Drosophila) 6.7 12q24.33 Receptor for the wingless type mouse mammary tumor virus (MMTV) integration site family 10631 POSTN Periostin, osteoblast-specific factor 6.7 13q13.3 Promotes integrin-dependent cell adhesion and motility, involved in extracellular matrix deposition

(Continued on the following page)

Cancer Res 2007; 67: (4).February 15, 2007 1760 www.aacrjournals.org

Table 1. Genes up-regulated by z6-fold in the tumor-associated endothelium (Cont’d)

Entrez gene ID Gene Description Fold Chromosomal Function difference location

85477 SCIN Scinderin 6.6 7p21.3 Ca2+-dependent actin filament severing protein, regulates cortical actin network dynamics 27242 TNFRSF21 Tumor necrosis factor receptor 6.6 6p21.1–12.2 Unclear; may be related to superfamily, member 21 activation of nuclear factor nB (NF-nB) and mitogen-activated protein kinase 8 (MAPK8)/c-Jun

NH2-terminal kinase (JNK), induces cell apoptosis, involved in inflammation and immunoregulation 25891 DKFZP586-H2123 Regeneration-associated muscle 6.2 11p13 Unknown protease, transcript variant 2 4582 MUC1 Mucin 1, transmembrane, 6.2 1q21 Regulates cell aggregation; transcript variant 4 adhesion 79084 MEP50 WD repeat domain 77, WDR77 6.1 1p13.2 Involved in the methylation and assembly of spliceosomal snRNAs Sm proteins 50509 COL5A3 Collagen, type V, a3 6.1 19p13.2 Extracellular protein, associated with formation of fibrils and some connective tissue pathology, such as inflammation, cancer, and atherosclerosis 6205 RPS11 Ribosomal protein S11 6.1 19q13.3 Involved in the recognition of termination codons 55803 CENTA2 Centaurin, a2 6.1 17q11.2 A phosphatidylinositide-binding protein present in the dense membrane fractions of cell extracts 2295 FOXF2 Forkhead box F2 6.0 6p25.3 Regulates cell proliferation and survival, associated with BMP and Wnt signaling

of digitized images to determine the thread length of the capillary-like (Supplementary Table S1). In addition, global analysis of the gene À network using a commercially available image analysis program (Northern list returned a P value of <5 Â 10 4, indicating that tumor and Eclipse, North Tonawanda, NY). endothelial isolates possess distinct expression profiles. Genes up-regulated in ovarian cancer endothelium. Of the 652 genes that were up-regulated z2-fold, 35 genes were elevated at Results least 6-fold in tumor endothelium (Table 1), with 9 being elevated Endothelial isolation and confirmation of cell purity. Five >10-fold. Multiple genes encoding proteins, such as collagens, normal ovaries and 10 invasive epithelial ovarian cancers were involved in extracellular matrix function, TNFAIP6, ADAMTS4, obtained and subjected to negative and positive immunoselection. MMP9, and MMP11, had increased expression in tumor vasculature Before carrying out microarray analysis, we tested the purity of all compared with normal ovarian vasculature. As expected, the av samples for endothelial cells with the endothelial cell markers integrin (vitronectin receptor) was elevated 2.5-fold in tumor P1H12 and von Willebrand factor. Immunostaining revealed that endothelium. Most of these genes have previously been shown to the immunopurification technique yielded endothelial cell purity of have increased expression in colon and breast cancer vasculature >95% in all samples (Fig. 1A). Thus, our isolation technique resulted (7, 8) and may reflect gene alterations regardless of tumor type. in a highly pure population of endothelial cells for subsequent Several transcription factors were up-regulated in the ovarian analyses. cancer vasculature. For example, HEYL was increased 3-fold, which Class comparison analysis of endothelial cell isolates. Total is also elevated in breast and colon cancer endothelium (7, 8). In RNA from purified endothelial cells was subjected to microarray addition, several novel transcription factors were identified, analysis using the Affymetrix Human U133 Plus 2.0 GeneChip including E2F transcription factor 3 (E2F3; plays a role in cell platform. A multivariate permutation t test (P < 0.001), providing proliferation; ref. 16), runt-related transcription factor 1 (RUNX1; has 95% confidence that the number of false discoveries did not exceed a direct role in angiogenesis; ref. 17), signal transducer and activator 10% of the complete gene list, identified 1,149 genes that were of transcription 2 (STAT2; involved in cellular proliferation; ref. 18), differentially regulated z2-fold in endothelium derived from the SNAIL-related zinc-finger transcription factor SLUG (SNAI2; epithelial ovarian cancers compared with normal ovarian tissue ref. 19), and Twist1 (20). These genes were elevated 2- to 18-fold in www.aacrjournals.org 1761 Cancer Res 2007; 67: (4).February 15, 2007

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research the ovarian cancer vasculature relative to normal ovarian endothe- Fyn (4.7-fold increase), which are known to play functionally lial cells. Whereas some of these genes have previously been shown significant roles for tumor cells and endothelial cells, may represent to have direct or indirect effects on angiogenesis, the functional novel targets for antivascular approaches (15). significance of others remains to be shown. Genes down-regulated in ovarian cancer endothelium. The Whereas vascular endothelial growth factor (VEGF)–based reduction of gene expression in tumor versus normal vasculature targeting has improved response rates to therapy and overall may reveal genes that function to suppress tumor and/or vascular survival in many cancers, most patients still eventually die from growth. Therefore, we next identified genes that were down- cancer. Therefore, additional targets are likely to be required to regulated in endothelial cells derived from ovarian cancer tissue. achieve curative therapy. Genes that are overexpressed on both There were 497 genes with z2-fold decrease in expression in tumor cells and tumor-associated endothelial cells may be tumor endothelium, with 17 decreased at least 6-fold (Table 2). particularly appealing as targets for antivascular therapy due to Interestingly, monoamine oxidase B (MAOB), a gene responsible for their ability to target both the epithelial and stromal compart- detoxification and degradation of monoamines, was decreased by ments. For example, epidermal growth factor receptor (EGFR) 6.4-fold in the tumor endothelial cells (22). Decorin, a small expression was increased by 3.5-fold in the tumor endothelium. multifunctional proteoglycan with antiangiogenic properties, was EGFR is known to be overexpressed in ovarian carcinomas and is decreased by 4.8-fold (23). Several other genes with potential predictive of poor outcome (21). We have previously shown that antiangiogenic or antiproliferative roles, such as fibulin-5 (FBLN-5) EGFR is overexpressed and phosphorylated in tumor endothelial and checkpoint suppressor 1 (CHES1), were down-regulated by cells, and dual targeting of VEGF receptor and EGFR in 4.5- and 4.3-fold, respectively (24, 25). The functional role of these combination with paclitaxel is highly efficacious (6). Similarly, and other down-regulated genes in the context of tumor angio- nonreceptor kinases such as FAK (or PTK2; 3.1-fold increase) and genesis remains to be determined.

Table 2. Down-regulated vascular genes in invasive epithelial ovarian cancer

Entrez gene ID Gene Description Fold Chromosomal Function difference location

5350 PLN Phospholamban 0.108 6q22.1 Inhibits sarcoplasmic reticulum Ca2+-ATPase activity 6401 SELE Selectin E, endothelial 0.112 1q22–q25 Cell-surface lycoprotein; inhibits adhesion molecule 1 cell adhesion; early marker of inflammation 9687 GREB1 GREB1 protein, transcript variant a 0.116 2p25.1 Transcription factor; inhibits cell proliferation 4969 OGN Osteoglycin osteoinductive factor, 0.147 9q22 Regulates collagen fibrillogenesis mimecan), transcript variant 3 25890 AB13BP ABI gene family, member 3 0.152 3q12 May play a role in cell motility by (NESH) binding protein regulating NESHfunction 90161 HS6ST2 Heparan sulfate 0.153 Xq26.2 Plays a role in growth factor 6-O-sulfotransferase 2 signaling, cell adhesion, and enzymatic catalysis; may be involved in vascularization by mediating fibroblast growth factor (FGF) signaling 139221 MUM1L1 Melanoma-associated antigen 0.155 Xq22.3 Encodes tumor-specific antigens (mutated) 1–like 1 4129 MAOB Monoamine oxidase B 0.156 Xp11.23 Regulates neurotransmitters in central nervous system 9452 ITM2A Integral membrane protein 2A 0.156 Xq13.3–Xq21.2 Transmembrane protein; marker of early-stage endochondral ossification 170302 ARX Aristaless related homeobox 0.162 Xp22.1–p21.3 Organ development; bifunctional transcription factor 10659 CUGBP2 CUG triplet repeat, RNA binding 0.163 10p13 Binds and stabilizes cyclooxygenase-2 protein 2, transcript variant 2 mRNA, inhibits its translation 5577 PRKAR2B Protein kinase, cyclic AMP (cAMP)– 0.163 7q22 Encodes a regulatory subunit RIIh dependent regulatory, type II, h of human cAMP-dependent protein kinase A 345557 LCXD3 Phosphatidylinositol-specific 0.165 5p13.1 Quantitatively solubilizes AChE phospholipase C, X domain from purified synaptic plasma containing 3 membranes and intact synaptosomes of Torpedo ocellata electric organ

Cancer Res 2007; 67: (4).February 15, 2007 1762 www.aacrjournals.org

Genes determined to be potential tumor endothelial To test the biological significance of some of these genes, we selected markers. We identified differentially regulated genes uniquely three genes: the novel polycomb group protein enhancer of Zeste expressed in tumor-associated endothelium versus normal endo- homologue 2 (EZH2), the notch receptor ligand Jagged1, and PTK2. thelium, and compared these to tumor-associated epithelial cells EZH2 plays an important role in many biological processes and is versus ovarian surface epithelium as potential endothelial-specific downstream of Akt activation (26), making it a potential antiangio- markers. We have previously reported expression profiling of genic target. SiRNA was used to silence EZH2 expression (Fig. 3A)in microdissected papillary serous ovarian cancers using the same HUVECs and its effects on migration and tube formation were microarray methods (10). The current list of differentially expressed examined. In comparison with control nonsilencing siRNA, EZH2 genes in tumor-associated endothelial cells was compared with the silencing resulted in a 85% decrease in endothelial tube formation gene list identified for laser microdissected tumor epithelial cells. on Matrigel (Fig. 3B). EZH2-targeted siRNA completely blocked A total of 534 differentially regulated genes were uniquely altered VEGF-stimulated migration of HUVECs (Fig. 3C). Similarly, to (up- or down-regulated) in the endothelial cells. The 28 genes with determine the functional relevance of Jagged1 (27) for endothelial the greatest (z6-fold) level of increase in the tumor related cell function, we tested the effects of Jagged1 silencing with siRNA endothelial cells are listed in Table 3. (Fig. 3A) on migration and tube formation. Jagged1-targeted siRNA Validation of gene expression alterations. The 23 validated reduced tube formation by 80% (Fig. 3B) and blocked VEGF- genes were selected in two rounds. To show the reproducibility of stimulated HUVEC migration (Fig. 3C). Similar results were noted the microarray analysis, a series of 17 genes were selected at with PTK2 silencing with siRNA (Fig. 3A–C). These data indicate that random, spanning a range of fold-changes (3.6–155.3; Fig. 1B). Of 17 the novel differentially expressed genes in the tumor-associated primer sets, 15 yielded specific quantitative real-time PCR products endothelial cells play functionally significant roles in angiogenesis. when analyzed using Universal Human Reference RNA (Stratagene, La Jolla, CA), with 13 reaching statistical significance in tumor (n = 10) and normal (n = 5) isolates (P < 0.05) including PLXDC1, Discussion ARRB2, HES4, PGF, EGFL6, ADAM12, COL5A3, COL18A1, PCOLCE, The major finding of the present study is that ovarian cancer– PMAIP1, CENTA2, TMEPAI, and NPTX2. To substantiate the path- associated endothelial cells contain a large number of gene way analysis (presented below), a second set of genes implicated alterations in comparison with normal ovarian endothelial cells. in endothelial tumor cell signaling was assessed. From a series We identified genes that are unique to the tumor vasculature and of 12 genes, suitable primer sets were obtained for 10 genes. All those that overlap in expression with tumor cells. To the best of our 10 pathway members were successfully validated (P < 0.05) includ- knowledge, this is the largest study of genomic profiling of ovarian ing FYN, VAV2, ECGF1, PTK2, TNFAIP6, EZH2, STC1, MMP9, JAG1, cancer–associated endothelial cells. and CSPG2. These data provide important confirmation of the Cancer is a heterogeneous disease that requires multimodality gene expression alterations identified by the microarray analysis. therapy. To date, most of the therapeutic approaches for ovarian To further examine whether the gene expression alterations cancer have focused on chemotherapy, which primarily targets identified by the microarray analysis also occur at the protein level, proliferating tumor cells. However, despite initial responses, most we did immunohistochemical staining for selected proteins on five tumors eventually develop resistance. Over the last few years, the normal ovaries and five invasive epithelial ovarian cancers. The critical role of the microenvironment in ovarian cancer growth and microarray analysis identified FAK (PTK2; 3.1-fold), Fyn (4.7-fold), progression has been established (6, 28, 29). The presumed genetic MMP9 (9.4-fold), b2-arrestin (4.8-fold), Jagged1 (4.3-fold), and stability of the microenvironment components such as endothelial PLXDC1 (10.2-fold) as being significantly increased in tumor- cells makes them an attractive therapeutic target. Indeed, associated endothelial cells, and these changes were validated biological therapies aimed at the microenvironment, such as by real-time RT-PCR (Fig. 1). Immunohistochemical peroxidase bevacizumab and VEGF Trap, are starting to show promise in staining confirmed that both FAK and Fyn were indeed overex- clinical trials (4) and preclinical models (30, 31) in ovarian and pressed in the tumor-associated endothelial cells (Fig. 1C and D)in other cancers. However, it is likely that additional targets will be all samples. There were no obvious differences in protein required to achieve further gains in therapeutic benefit. Genomic expression between arterioles and venules. Similarly, increased profiling of tumor-associated endothelial cells, as described in the expression of MMP9, h2-arrestin, Jagged1, and PLXDC1 was also current study, is a powerful method for identifying novel genes that confirmed at the protein level (Fig. 1C and D). may be potential targets or biomarkers. Signaling pathways modulated in tumor endothelium and It has become evident that endothelial cells vary phenotypically their functional significance. Ovarian epithelial carcinomas arise depending on the organ of origin (32). To date, there are limited from molecular events occurring in the epithelial layer, which affect data about genomic differences in tumor-associated endothelial changes in gene expression within surrounding nonepithelial cell cells from specific tumors. A small number of either colon, breast, populations. For endothelial cells, this altered signaling environment or brain tumors have been evaluated previously using serial stimulates proliferation, migration, and tumor vascularization. To analysis of gene expression (7–9). Whereas there was overlap in the identify epithelial genes that may be responsible for these changes gene profile of endothelial cells from ovarian versus these other and the endothelial signaling pathways that are affected, a series of cancers, there were also clear differences. For example, genes such laser microdissected papillary serous epithelial cell isolates and as av-integrin, ADAMTS4, HEYL, and MMP9 were increased in ovarian surface epithelial brushings were compared, as previously ovarian cancer vasculature and have been noted to have increased described (10). Pathway diagrams were generated using PathwayAs- expression in the vasculature from other cancers as well. However, sist version 3.0 software (Fig. 2A and B). The genes comprising the several unique genes such as TNFAIP6, E2F3, EZH2, and RUNX1 pathway suggest involvement in endothelial cell proliferation, tube were also discovered. Whether these are indeed unique to ovarian formation, and cell motility. The genes in this pathway are cancer vasculature or tumor vasculature more broadly will require summarized in Fig. 2C, along with the inferred biological function. further investigation. www.aacrjournals.org 1763 Cancer Res 2007; 67: (4).February 15, 2007

Table 3. Genes specifically regulated in tumor endothelium

Entrez gene ID Gene Description Fold Chromosomal Function difference location

25975 EGFL6 EGF-like domain, multiple 6 36.848 Xp22 May regulate cell cycle and oncogenesis 7130 TNFAIP6 Tumor necrosis factor, a-induced 29.062 2q23.3 Anti-inflammatory and protein 6 chondroprotective effect 7291 TWIST1 Twist homologue 1 18.969 7p21.2 Inhibits chondrogenesis (acrocephalosyndactyly 3; Saethre-Chotzen syndrome; Drosophila) 6781 STC1 Stanniocalcin 1 13.326 8p21–p11.2 Regulates calcium/phosphate homeostasis and cell metabolism 84525 HOP Homeodomain-only protein, 13.144 4q11–q12 Transcriptional repressor; transcript variant 2 modulates serum response factor–dependent cardiac- specific gene expression and cardiac development 1462 CSPG2 Chondroitin sulfate proteoglycan 2 10.355 5q14.3 Extracellular matrix component (versican) of the vitreous gel; anti–cell adhesive 57125 PLXDC1 Plexin domain containing 1 10.215 17q21.1 Unknown 4318 MMP9 Matrix metallopeptidase 9 9.389 20q11.2–q13.1 Breakdown of extracellular (gelatinase B, 92-kDa matrix; plays a role in gelatinase, 92-kDa angiogenesis and type IV collagenase) tumor invasion 3937 LCP2 Lymphocyte cytosolic protein 2 8.744 5q33.1–qter Promotes T-cell development (SH2 domain containing and activation leukocyte protein of 76 kDa) 5366 PMAIP1 Phorbol-12-myristate-13-acetate- 8.543 18q21.32 Unknown induced protein 1 8038 ADAM12 ADAM metallopeptidase domain 12 7.605 10q26.3 Involved in cell-cell and (meltrin a), transcript variant 1 cell-matrix interactions 25878 MXRA5 Matrix-remodeling associated 5 6.865 Xp22.33 Matrix remodeling 1123 CHN1 Chimerin (chimaerin) 1 6.857 2q31–q32.1 Rho GTPase activating protein 3310 HSPA6 Heat shock 70-kDa protein 6 6.76 1q23 Involved in protein conformational interactions 10631 POSTN Periostin, osteoblast-specific factor 6.732 13q13.3 Promotes integrin-dependent cell adhesion and motility; involved in extracellular matrix deposition 11211 FZD10 Frizzled homologue 10 (Drosophila) 6.701 12q24.33 Receptor for the wingless type MMTV integration site family 27242 TNFRSF21 Tumor necrosis factor receptor 6.649 6p21.1–12.2 Activates NF-nB and MAPK8/JNK, superfamily, member 21 induces cell apoptosis, involved in inflammation and immunoregulation 25891 DKFZP586H2123 Regeneration-associated muscle 6.199 11p13 Unknown protease, transcript variant 2 79084 MEP50 WD repeat domain 77 6.144 1p13.2 Involved in the methylation and assembly of spliceosomal snRNAs Sm proteins 50509 COL5A3 Collagen, type V, a3 6.118 19p13.2 Extracellular protein, associated with formation of fibrils and some connective tissue pathology, such as inflammation, cancer, and atherosclerosis 6205 RPS11 Ribosomal protein S11 6.095 19q13.3 Involved in the recognition of termination codons

(Continued on the following page)

Cancer Res 2007; 67: (4).February 15, 2007 1764 www.aacrjournals.org

Table 3. Genes specifically regulated in tumor endothelium (Cont’d)

Entrez gene ID Gene Description Fold Chromosomal Function difference location

55803 CENTA2 Centaurin, a2 6.09 17q11.2 A phosphatidylinositide- binding protein present in the dense membrane fractions of cell extracts 90161 HS6ST2 Heparan sulfate 6-O-sulfotransferase 2 0.153 Xq26.2 Plays a role in growth factor signaling, cell adhesion, and enzymatic catalysis; may be involved in vascularization by mediating FGF signaling 4969 OGN Osteoglycin (osteoinductive factor, 0.147 9q22 Regulates collagen fibrillogenesis mimecan), transcript variant 3 9687 GREB1 GREB1 protein, transcript variant a 0.116 2p25.1 Transcription factor; inhibits cell proliferation 6401 SELE Selectin E (endothelial 0.112 1q22–q25 Cell-surface lycoprotein; inhibits adhesion molecule 1) cell adhesion; early marker of inflammation 5350 PLN Phospholamban 0.108 6q22.1 Inhibits sarcoplasmic reticulum Ca2+-ATPase activity

A number of genes that were identified are known to play other cancers (41). To the best of our knowledge, this is the functionally significant roles in both tumor cells and tumor- first report that EZH2 is overexpressed in tumor-associated associated endothelial cells. For example, PTK2 (FAK) has been endothelial cells. In the present study, the microarray data yielded shown to play a role in ovarian cancer cell migration and a 2.9-fold higher EZH2 expression in ovarian cancer–related invasion (15) and survival (33). With regard to endothelial cells, endothelium compared with normal ovary. We also showed the PTK2 plays a pivotal role in angiogenesis related to late functional relevance of EZH2 for endothelial cell migration and embryonic development (34) and modulates endothelial cell tube formation using RNA interference. HUVECs were used for migration (35). Data from our study show that PTK2 expression these assays and it is possible that the role of EZH2 may be was up-regulated in tumor-related endothelium. We have recently different in ovarian or mesenteric endothelial cells. Whether shown that PTK2 silencing with siRNA sensitized tumor cells to EZH2 is a valid antiangiogenic target will require further investi- docetaxel chemotherapy in vitro (33). Moreover, in vivo FAK gation, but its role in endothelial function combined with its silencing with siRNA incorporated in neutral liposomes was significance for tumor cell function makes it an appealing highly efficacious in both chemotherapy-sensitive and chemo- candidate. therapy-resistant ovarian cancer models through both direct and Pathway analysis identified coordinated signaling events stim- indirect antitumor effects by decreasing VEGF levels (36). Based ulated by transformed ovarian epithelial cells, which may be on the results of the current study, we anticipate that such effects modulating tumor endothelial cell behavior. The proangiogenic may be even greater when FAK is suppressed directly in effect of EZH2 on endothelial cells and increased CCNE1 levels endothelial cells. may be associated, in part, with epithelial cell–induced VEGF It is well known that Src family tyrosine kinases play an signaling. In cortical neuron precursors, VEGF induces E2F3 essential role in the signaling of integrin-mediated biological expression (42). Whereas neuron development and migration is processes such as cell proliferation, differentiation, actin organi- seemingly unrelated to angiogenesis, there is increasing evidence zation, and cell migration (37). The Src family member Fyn plays that the two processes may use analogous pathways (43). a key role in endothelial cell signaling pathways resulting in Enhanced E2F3 expression may result in elevated levels of EZH2 stimulation of endothelial migration and tube formation (38). In and CCN1, both of which are direct targets of the transcrip- the present study, gene profiling revealed that Fyn expression was tion factor (44, 45). Notch signaling has also been linked to increased by 4.7-fold in tumor-related endothelium, and this the regulation of tube formation and the ability of endothelial increase was validated at both mRNA and protein levels. Based on cells to establish a mature phenotype (46). Both JAG2, secreted its known role in endothelial cell function and significant up- by epithelial tumor cells, and endothelial cell–derived JAG1 can regulation in tumor-associated endothelial cells, Fyn may be an activate the Notch pathway. HESR1 is a Notch responsive antiangiogenic target. transcription factor that has been implicated in the regulation One of the novel genes identified from the current study is of endothelial cell tube formation (47). Consequently, the ability EZH2, a member of the polycomb group of genes (PcG), which of JAG1 to stimulate tube formation may be mediated through are important for transcriptional regulation through nucleo- the downstream targets of this gene. In addition to promoting some modification, chromatin remodeling, and interaction with endothelial cell tube formation and proliferation, secreted other transcription factors (39). EZH2 is controlled by E2F and is epithelial factors may also drive endothelial cell motility. For also involved in p53-regulated cell cycle control (40). EZH2 was example, SPP1 can engage the av integrin receptor and support previously shown to be overexpressed in prostate, breast, and directional cell migration (48). Furthermore, it can interact www.aacrjournals.org 1765 Cancer Res 2007; 67: (4).February 15, 2007

Cancer Res 2007; 67: (4).February 15, 2007 1766 www.aacrjournals.org

Figure 3. A, siRNA-mediated silencing of EZH2, Jagged1, and PTK2 was assessed using immunofluorescence and Western blot. B, effect of EZH2, Jagged1,orPTK2 silencing on HUVEC tube formation. C, effect of EZH2, Jagged1,orPTK2 silencing on HUVEC migration. *, P <0.01.

with the CD44 receptor stimulating VAV2 activity (49). This in processes related to tumor vascularization, invasion, and interaction may be stabilized by TNFAIP6, resulting in CD44 metastatic growth. Some of these may offer opportunities for receptor clustering and enhanced signaling (50). The net effect therapeutic intervention. of these interactions is increased FAK stimulation and endothelial cell migration. Signaling events originating from epithelial tumor cells and their downstream endothelial effectors Acknowledgments represent a reservoir of putative targets for therapeutic interven- Received 10/10/2006; revised 11/26/2006; accepted 12/4/2006. tion. Of particular interest are the novel genes E2F3, EZH2, and Grant support: NIHgrants CA10929801 and CA11079301, Program Project TNFAIP6, which may directly participate in critical proangiogenic Development Grant from the Ovarian Cancer Research Fund, The Marcus Foundation, The Zarrow Foundation, and the University of Texas M. D. Anderson Cancer Center pathways. Specialized Program of Research Excellence in Ovarian Cancer grant P50 CA 083639. In summary, the expression profile of ovarian cancer–associated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance endothelial cells is distinct and unique. However, there are with 18 U.S.C. Section 1734 solely to indicate this fact. similarities with tumor vasculature in other organs. Moreover, We thank Drs. Isaiah J. Fidler and Robert Langley at the University of Texas M. D. there are multiple genes that have increased expression in both Anderson Cancer Center (Houston, TX) for helpful input and discussions about this work; Joseph Celestino for assistance with specimen collection; Donna Reynolds for tumor-associated endothelial cells and tumor cells. Additional assistance with immunohistochemistry; and Susan Davis and Catherine Rodgers for work is needed to define the role of the novel genes identified here assistance with manuscript preparation.

Figure 2. Analysis of putative pathways stimulated in tumor endothelial cells by papillary serous ovarian epithelial tumor cells. Pathway diagrams were generated with the assistance of PathwayAssist version 3.0software using gene expression data. Genes included in the pathway were required to have a fold change val ue of z2.0. For some genes, multiple probe sets were averaged. A, differentially expressed genes modulated in tumor endothelial cells and their associated interactions are described. Green oval, gene is up-regulated in endothelial tumor samples; red oval, down-regulation; gray oval, genes displaying no change in expression. Green ovals with a red border, secreted genes up-regulated in ovarian epithelial tumor cells. B, pathways implicated in cell proliferation, tube formation (JAG1, JAG2, NOTCH1, HESR1, EZH2, and STC1), and cell motility. C, table describing pathway genes and their inferred biological function. www.aacrjournals.org 1767 Cancer Res 2007; 67: (4).February 15, 2007

References 19. Perez-Mancera PA, Gonzalez-Herrero I, Perez-Caro 35. Kaczmarek E, Erb L, Koziak K, et al. Modulation of M, et al. SLUG in cancer development. Oncogene 2005; endothelial cell migration by extracellular nucleotides: 1. Cannistra SA. Cancer of the ovary. N Engl J Med 1993; 24:3073–82. involvement of focal adhesion kinase and phosphatidy- 329:1550–9. 20. Mironchik Y, Winnard PT, Jr., Vesuna F, et al. Twist linositol 3-kinase-mediated pathways. Thromb Haemost 2. Folkman J. What is the evidence that tumors are overexpression induces in vivo angiogenesis and corre- 2005;93:735–42. angiogenesis dependent? J Natl Cancer Inst 1990;82:4–6. lates with chromosomal instability in breast cancer. 36. Halder J, Kamat AA, Landen CN, et al. Focal adhesion 3. Hurwitz H, Fehrenbacher L, Novotny W, et al. Cancer Res 2005;65:10801–9. kinase targeting using in vivo short interfering RNA Bevacizumab plus irinotecan, fluorouracil, and leuco- 21. Berchuck A, Rodriguez GC, Kamel A, et al. delivery in neutral liposomes for ovarian carcinoma vorin for metastatic colorectal cancer. N Engl J Med Epidermal growth factor receptor expression in normal therapy. Clin Cancer Res 2006;12:4916–24. 2004;350:2335–42. ovarian epithelium and ovarian cancer. I. Correlation 37. Playford MP, Schaller MD. The interplay between Src 4. Jain RK, Duda DG, Clark JW, Loeffler JS. Lessons from of receptor expression with prognostic factors in and integrins in normal and tumor biology. Oncogene phase III clinical trials on anti-VEGF therapy for cancer. patients with ovarian cancer. Am J Obstet Gynecol 2004;23:7928–46. Nat Clin Pract Oncol 2006;3:24–40. 1991;164:669–74. 38. Mochizuki Y, Nakamura T, Kanetake H, Kanda S. 5. Cooper BC, Ritchie JM, Broghammer CL, et al. 22. Grimsby J, Toth M, Chen K, et al. Increased stress Angiopoietin2stimulatesmigrationandtube-like Preoperative serum vascular endothelial growth factor response and h-phenylethylamine in MAOB-deficient structure formation of murine brain capillary endothe- levels: significance in ovarian cancer. Clin Cancer Res mice. Nat Genet 1997;17:206–10. lial cells through c-Fes and c-Fyn. J Cell Sci 2002;115: 2002;8:3193–7. 23. Sulochana KN, Fan H, Jois S, et al. Peptides derived 175–83. 6. Thaker PH, Yazici S, Nilsson MB, et al. Antivascular from human decorin leucine-rich repeat 5 inhibit 39. Varambally S, Dhanasekaran SM, Zhou M, et al. The therapy for orthotopic human ovarian carcinoma angiogenesis. J Biol Chem 2005;280:27935–48. polycomb group protein EZH2 is involved in progres- through blockade of the vascular endothelial growth 24. Albig AR, Schiemann WP. Fibulin-5 antagonizes sion of prostate cancer. Nature 2002;419:624–9. factor and epidermal growth factor receptors. Clin vascular endothelial growth factor (VEGF) signaling 40. Tang X, Milyavsky M, Shats I, Erez N, Goldfinger N, Cancer Res 2005;11:4923–33. and angiogenic sprouting by endothelial cells. DNA Cell Rotter V. Activated p53 suppresses the histone 7. Parker BS, Argani P, Cook BP, et al. Alterations in Biol 2004;23:367–9. methyltransferase EZH2 gene. Oncogene 2004;23: vascular gene expression in invasive breast carcinoma. 25. Scott KL, Plon SE. CHES1/FOXN3 interacts with Ski- 5759–69. Cancer Res 2004;64:7857–66. interacting protein and acts as a transcriptional 41. Kleer CG, Cao Q, Varambally S, et al. EZH2 is a 8. St Croix B, Rago C, Velculescu V, et al. Genes repressor. Gene 2005;359:119–26. marker of aggressive breast cancer and promotes expressed in human tumor endothelium. Science 2000; 26. Cha TL, Zhou BP, Xia W, et al. Akt-mediated neoplastic transformation of breast epithelial cells. Proc 289:1197–202. phosphorylation of EZH2 suppresses methylation of Natl Acad Sci U S A 2003;100:11606–11. 9. Madden SL, Cook BP, Nacht M, et al. Vascular gene lysine 27 in histone H3. Science 2005;310:306–10. 42. Zhu Y, Jin K, Mao XO, Greenberg DA. Vascular expression in nonneoplastic and malignant brain. Am J 27. Zeng Q, Li S, Chepeha DB, et al. Crosstalk between endothelial growth factor promotes proliferation of Pathol 2004;165:601–8. tumor and endothelial cells promotes tumor angiogen- cortical neuron precursors by regulating E2F expression. 10. Bonome T, Lee JY, Park DC, et al. Expression profiling esis by MAPK activation of Notch signaling. Cancer Cell FASEB J 2003;17:186–93. of serous low malignant potential, low-grade, and high- 2005;8:13–23. 43. Carmeliet P. Blood vessels and nerves: common grade tumors of the ovary. Cancer Res 2005;65:10602–12. 28. Sood AK, Seftor EA, Fletcher MS, et al. Molecular signals, pathways and diseases. Nat Rev Genet 2003;4: 11. Simon R, McShane L, Radmacher M, Wright G, Zhao determinants of ovarian cancer plasticity. Am J Pathol 710–20. Y. Design and analysis of DNA microarray investiga- 2001;158:1279–88. 44. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, tions. New York: Springer-Verlag; 2004. 29. Liotta LA, Kohn EC. The microenvironment of the Helin K. EZH2 is downstream of the pRB-E2F pathway, 12. Wright GW, Simon RM. A random variance model for tumour-host interface. Nature 2001;411:375–9. essential for proliferation and amplified in cancer. detection of differential gene expression in small micro- 30. Hu L, Hofmann J, Holash J, Yancopoulos GD, Sood EMBO J 2003;22:5323–35. array experiments. Bioinformatics 2003;19:2448–55. AK, Jaffe RB. Vascular endothelial growth factor trap 45. Humbert PO, Verona R, Trimarchi JM, Rogers C, 13. Donninger H, Bonome T, Radonovich M, et al. Whole combined with paclitaxel strikingly inhibits tumor and Dandapani S, Lees JA. E2f3 is critical for normal cellular genome expression profiling of advance stage papillary ascites, prolonging survival in a human ovarian cancer proliferation. Genes Dev 2000;14:690–703. serous ovarian cancer reveals activated pathways. model. Clin Cancer Res 2005;11:6966–71. 46. Sainson RC, Harris AL. Hypoxia-regulated differenti- Oncogene 2004;23:8065–77. 31. Byrne AT, Ross L, Holash J, et al. Vascular endo- ation: let’s step it up a Notch. Trends Mol Med 2006;12: 14. Livak KJ, Schmittgen TD. Analysis of relative gene thelial growth factor-trap decreases tumor burden, 141–3. expression data using real-time quantitative PCR and inhibits ascites, and causes dramatic vascular remodel- 47. Henderson AM, Wang SJ, Taylor AC, Aitkenhead M, the 2(ÀDDC(T)) method. Methods 2001;25:402–8. ing in an ovarian cancer model. Clin Cancer Res 2003;9: Hughes CC. The basic helix-loop-helix transcription 15. Sood AK, Coffin JE, Schneider GB, et al. Biological 5721–8. factor HESR1 regulates endothelial cell tube formation. significance of focal adhesion kinase in ovarian cancer: 32. Langley RR, Ramirez KM, Tsan RZ, Van Arsdall M, J Biol Chem 2001;276:6169–76. role in migration and invasion. Am J Pathol 2004;165: Nilsson MB, Fidler IJ. Tissue-specific microvascular 48. Liaw L, Skinner MP, Raines EW, et al. The adhesive 1087–95. endothelial cell lines from H-2K(b)-tsA58 mice for and migratory effects of osteopontin are mediated via 16. Black EP, Hallstrom T, Dressman HK, West M, Nevins studies of angiogenesis and metastasis. Cancer Res distinct cell surface integrins. Role of avh3 in smooth JR. Distinctions in the specificity of E2F function 2003;63:2971–6. muscle cell migration to osteopontin in vitro. J Clin revealed by gene expression signatures. Proc Natl Acad 33. Halder J, Landen CN, Jr., Lutgendorf SK, et al. Focal Invest 1995;95:713–24. Sci U S A 2005;102:15948–53. adhesion kinase silencing augments docetaxel-mediated 49. Lin YH, Yang-Yen HF. The osteopontin-CD44 survival 17. Iwatsuki K, Tanaka K, Kaneko T, et al. Runx1 promotes apoptosis in ovarian cancer cells. Clin Cancer Res 2005; signal involves activation of the phosphatidylinositol 3- angiogenesis by down-regulation of insulin-like growth 11:8829–36. kinase/Akt signaling pathway. J Biol Chem 2001;276: factor-binding protein-3. Oncogene 2005;24:1129–37. 34. Shen TL, Park AY, Alcaraz A, et al. Conditional 46024–30. 18. Gomez D, Reich NC. Stimulation of primary human knockout of focal adhesion kinase in endothelial cells 50. Lesley J, Gal I, Mahoney DJ, et al. TSG-6 modulates endothelial cell proliferation by IFN. J Immunol 2003; reveals its role in angiogenesis and vascular develop- the interaction between hyaluronan and cell surface 170:5373–81. ment in late embryogenesis. J Cell Biol 2005;169:941–52. CD44. J Biol Chem 2004;279:25745–54.

Cancer Res 2007; 67: (4).February 15, 2007 1768 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Gene Alterations Identified by Expression Profiling in Tumor-Associated Endothelial Cells from Invasive Ovarian Carcinoma

Chunhua Lu, Tomas Bonome, Yang Li, et al.

Cancer Res 2007;67:1757-1768.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/67/4/1757

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2007/02/14/67.4.1757.DC1

Cited articles This article cites 49 articles, 23 of which you can access for free at: http://cancerres.aacrjournals.org/content/67/4/1757.full#ref-list-1

Citing articles This article has been cited by 30 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/67/4/1757.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/67/4/1757. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.