Novel report of expression and function of CD97 in malignant gliomas: correlation with Wilms tumor 1 expression and glioma cell invasiveness

Laboratory investigation

Restricted access


The Wilms tumor 1 (WT1) protein—a developmentally regulated transcription factor—is aberrantly expressed in gliomas and promotes their malignant phenotype. However, little is known about the molecular allies that help it mediate its oncogenic functions in glioma cells.


The authors used short interfering RNA (siRNA) to suppress WT1 expression in glioblastoma (GBM) cells and evaluated the effect of this on GBM cell invasiveness. Gene expression analysis was then used to identify the candidate genes that were altered as a result of WT1 silencing. One candidate target, CD97, was then selected for further investigation into its role by suppressing its expression using siRNA silencing, followed by proliferation and invasion assays.


WT1 levels were reliably and reproducibly suppressed by siRNA application. This resulted in a significant decrease in cellular invasiveness. Microarray analyses identified the gene products that were consistently downregulated (27) and upregulated (11) with WT1 silencing. Of these, CD97 expression was consistently suppressed across the 3 different GBM cell lines studied and was found on further investigation to significantly impact GBM cell invasiveness.


Although CD97 expression in gliomas has not been described previously, we conclude that the possible upregulation of CD97 mediated by WT1 promotes cellular invasiveness—one of the most characteristic and challenging aspects of glial tumor cells. Further studies are needed to clarify the nature of this regulation and its impact, as CD97 could represent a novel target for antiglioma therapies.

Abbreviations used in this paper:ATP = adenosine triphosphate; EGF = epidermal growth factor; GBM = glioblastoma; PDGF = platelet-derived growth factor; qRT-PCR = quantitative reverse transcriptase polymerase chain reaction; SDS = sodium dodecyl sulfate; siRNA = short interfering RNA; VCU = Virginia Commonwealth University; WT1 = Wilms tumor 1.

Article Information

Current affiliation for Dr. Van Meter: Pediatric Hematology-Oncology, Department of Pediatrics, VCU School of Medicine.

Address correspondence to: William C. Broaddus, M.D., Ph.D., Department of Neurosurgery, Virginia Commonwealth University, P.O. Box 980631, Richmond, Virginia 23298-0631. email:

Please include this information when citing this paper: published online February 7, 2012; DOI: 10.3171/2011.11.JNS111455.

© AANS, except where prohibited by US copyright law.



  • View in gallery

    Expression and function of WT1 in U1242-MG and GBM-6 cells. A: WT1 expression in GBM-6 and U1242-MG cells was detected using Western blotting. Twenty micrograms of protein was loaded in each lane. Cell lysates in Lanes A, B, and C are from PC3 (control), GBM-6, U1242-MG cells, respectively. B: WT1 silencing was confirmed using qRT-PCR (p ≤ 0.001). WT1 mRNA levels in anti-WT1 siRNA–treated cells are first normalized to the level of the loading control and expressed as a percentage of the control cells. **p ≤ 0.001. C: WT1 downregulation was associated with a decrease in GBM-6 and U1242-MG cellular invasiveness at Day 4 posttransfection (*p < 0.05, ***p < 0.001). Cell invasiveness in WT1-silenced cells was assessed by using the CellTiter-Glo assay (RLU is a surrogate for cell number) to first quantify the number of cells that had invaded through the Matrigel in the si groups versus the scr groups and then expressing this value as a percentage of that seen in control (scr) cells. All experiments were performed in triplicate with separate cultured samples of tumor cells treated in parallel with siRNA against WT1 and corresponding controls.

  • View in gallery

    Supervised cluster analysis. Two-dimensional hierarchical clustering of samples and genes using Pearson (centered) correlation and average linkage. Three independent transfection experiments were performed for each microarray analysis.

  • View in gallery

    Validation of microarray results. U251-MG cells were transfected with nontargeting siRNA (scr) or WT1-targeting siRNA (si). RNA samples extracted 48 hours following transfection were analyzed by qRT-PCR. Bar Graph: Fold-change values of siRNA-treated cells compared with scr controls (depicted by the dashed line at 1) calculated from qRT-PCR experiments. Line Graph: Fold-change values calculated using microarray analyses. Value of scr is set at 1 (depicted by the dashed line). Transfection was performed on 3 parallel sets of cell cultures to generate these results. *p ≤ 0.05; **p ≤ 0.01.

  • View in gallery

    Confirmation of altered regulation of target genes across different glioma cell lines transfected with anti-WT1 siRNA. Experiments were performed in triplicate for both GBM-6 and U1242-MG cells. The x-axes represent the target genes and the y-axes the fold change in si cells (value of scr set at 1, dashed line). *p ≤ 0.05; **p < 0.01. §§Genes that were not found to correlate/be expressed.

  • View in gallery

    Expression and function of CD97 in GBM cells. A: Western blot showing CD97 protein expression (≈ 92 kDa) in U251-MG, U1242-MG, and GBM-6 glioma cells. Ten micrograms of protein was loaded in each lane. Rabbit polyclonal Ab (Abcam) was used to detect CD97 (1:200). B: Quantitative RT-PCR demonstrated minimal expression of CD97 in normal human astrocytes (NHA) and a 6- to 21-fold increase in expression in the GBM cell lines investigated. C: Using siRNA directed against CD97, we confirmed a significant knockdown in CD97 RNA in the tumor cells on Day 4 posttransfection by qRT-PCR in U251-MG, U1242-MG, and GBM-6 cells. D: Treatment of U251-MG, U1242-MG, and GBM-6 cells with siRNA against CD97 resulted in a significant decrease in their ability to invade through the Matrigel-coated filters of transwell plates compared with cells that were treated with nontargeting siRNA. Cell invasiveness in CD97-silenced cells was expressed by using the CellTiter-Glo assay to first quantify the number of cells that had invaded through the Matrigel in the si groups versus scr groups and then expressing this value as a percentage of that seen in control (scr) cells. Three independent cultures of U251-MG, GBM-6, and U1242-MG cells were transfected with corresponding controls. *p < 0.05.



Aust GEichler WLaue SLehmann IHeldin NELotz O: CD97: a dedifferentiation marker in human thyroid carcinomas. Cancer Res 57:179818061997


Cheever MAAllison JPFerris ASFinn OJHastings BMHecht TT: The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res 15:532353372009


Chen MYClark AJChan DCWare JLHolt SEChidambaram A: Wilms' tumor 1 silencing decreases the viability and chemoresistance of glioblastoma cells in vitro: a potential role for IGF-1R de-repression. J Neurooncol 103:871022011


Clark AJChan DCChen MYFillmore HLDos Santos WGVan Meter TE: Down-regulation of Wilms' tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53. J Neurooncol 83:1631722007


Clark AJDos Santos WGMcCready JChen MYVan Meter TEWare JL: Wilms tumor 1 expression in malignant gliomas and correlation of +KTS isoforms with p53 status. J Neurosurg 107:5865922007


Clark AJWare JLChen MYGraf MRVan Meter TEDos Santos WG: Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells. Laboratory investigation. J Neurosurg 112:18252010


Dudnakova TSpraggon LSlight JHastie N: Actin: a novel interaction partner of WT1 influencing its cell dynamic properties. Oncogene 29:108510922010


Dumur CINasim SBest AMArcher KJLadd ACMas VR: Evaluation of quality-control criteria for microarray gene expression analysis. Clin Chem 50:199420022004


Dumur CISana SLadd ACFerreira-Gonzalez AWilkinson DSPowers CN: Assessing the impact of tissue devitalization time on genome-wide gene expression analysis in ovarian tumor samples. Diagn Mol Pathol 17:2002062008


Garrido-Ruiz MCRodriguez-Pinilla SMPérez-Gómez BRodriguez-Peralto JL: WT 1 expression in nevi and melanomas: a marker of melanocytic invasion into the dermis. J Cutan Pathol 37:5425482010


Gerald WLHaber DA: The EWS-WT1 gene fusion in desmoplastic small round cell tumor. Semin Cancer Biol 15:1972052005


Giannini CSarkaria JNSaito AUhm JHGalanis ECarlson BL: Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme. Neuro Oncol 7:1641762005


Gray JXHaino MRoth MJMaguire JEJensen PNYarme A: CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation. J Immunol 157:543854471996


Han SLXu CWu XLLi JLLiu ZZeng QQ: The impact of expressions of CD97 and its ligand CD55 at the invasion front on prognosis of rectal adenocarcinoma. Int J Colorectal Dis 25:6957022010


Hohenstein PHastie ND: The many facets of the Wilms' tumour gene, WT1. Hum Mol Genet 15 Spec No 2:R196R2012006


Huff V: Wilms' tumours: about tumour suppressor genes, an oncogene and a chameleon gene. Nat Rev Cancer 11:1111212011


Izumoto STsuboi AOka YSuzuki THashiba TKagawa N: Phase II clinical trial of Wilms tumor 1 peptide vaccination for patients with recurrent glioblastoma multiforme. J Neurosurg 108:9639712008


Jomgeow TOji YTsuji NIkeda YIto KTsuda A: Wilms' tumor gene WT1 17AA(−)/KTS(−) isoform induces morphological changes and promotes cell migration and invasion in vitro. Cancer Sci 97:2592702006


Kreidberg JASariola HLoring JMMaeda MPelletier JHousman D: WT-1 is required for early kidney development. Cell 74:6796911993


Kwakkenbos MJKop ENStacey MMatmati MGordon SLin HH: The EGF-TM7 family: a postgenomic view. Immunogenetics 55:6556662004


Liu YChen LPeng SChen ZGimm OFinke R: The expression of CD97EGF and its ligand CD55 on marginal epithelium is related to higher stage and depth of tumor invasion of gastric carcinomas. Oncol Rep 14:141314202005


Loeb DMEvron EPatel CBSharma PMNiranjan BBuluwela L: Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation. Cancer Res 61:9219252001


Mayo MWWang CYDrouin SSMadrid LVMarshall AFReed JC: WT1 modulates apoptosis by transcriptionally upregulating the bcl-2 proto-oncogene. EMBO J 18:399040031999


Miyagi TAhuja HKubota TKubonishi IKoeffler HPMiyoshi I: Expression of the candidate Wilm's tumor gene, WT1, in human leukemia cells. Leukemia 7:9709771993


Oji YInohara HNakazawa MNakano YAkahani SNakatsuka S: Overexpression of the Wilms' tumor gene WT1 in head and neck squamous cell carcinoma. Cancer Sci 94:5235292003


Oji YMiyoshi SMaeda HHayashi STamaki HNakatsuka S: Overexpression of the Wilms' tumor gene WT1 in de novo lung cancers. Int J Cancer 100:2973032002


Oji YMiyoshi YKoga SNakano YAndo ANakatsuka S: Overexpression of the Wilms' tumor gene WT1 in primary thyroid cancer. Cancer Sci 94:6066112003


Oji YNakamori SFujikawa MNakatsuka SYokota ATatsumi N: Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma. Cancer Sci 95:5835872004


Oji YOgawa HTamaki HOka YTsuboi AKim EH: Expression of the Wilms' tumor gene WT1 in solid tumors and its involvement in tumor cell growth. Jpn J Cancer Res 90:1942041999


Oji YYamamoto HNomura MNakano YIkeba ANakatsuka S: Overexpression of the Wilms' tumor gene WT1 in colorectal adenocarcinoma. Cancer Sci 94:7127172003


Oji YYano MNakano YAbeno SNakatsuka SIkeba A: Overexpression of the Wilms' tumor gene WT1 in esophageal cancer. Anticancer Res 24:310331082004


Oka YSugiyama H: WT1 peptide vaccine, one of the most promising cancer vaccines: its present status and the future prospects. Immunotherapy 2:5915942010


Oka YTsuboi AFujiki FLi ZNakajima HHosen N: WT1 peptide vaccine as a paradigm for “cancer antigen-derived peptide”-based immunotherapy for malignancies: successful induction of anti-cancer effect by vaccination with a single kind of WT1 peptide. Anticancer Agents Med Chem 9:7877972009


Perugorria MJCastillo JLatasa MUGoñi SSegura VSangro B: Wilms' tumor 1 gene expression in hepatocellular carcinoma promotes cell dedifferentiation and resistance to chemotherapy. Cancer Res 69:135813672009


Scharnhorst Vvan der Eb AJJochemsen AG: WT1 proteins: functions in growth and differentiation. Gene 273:1411612001


Scholz HWagner KDWagner N: Role of the Wilms' tumour transcription factor, Wt1, in blood vessel formation. Pflugers Arch 458:3153232009


Sugiyama H: WT1 (Wilms' tumor gene 1): biology and cancer immunotherapy. Jpn J Clin Oncol 40:3773872010


Tamaki HOgawa HOhyashiki KOhyashiki JHIwama HInoue K: The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia 13:3933991999


Ueda TOji YNaka NNakano YTakahashi EKoga S: Overexpression of the Wilms' tumor gene WT1 in human bone and soft-tissue sarcomas. Cancer Sci 94:2712762003


Van Meter TEDumur CIHafez NGarrett CFillmore HLBroaddus WC: Microarray analysis of MRI-defined tissue samples in glioblastoma reveals differences in regional expression of therapeutic targets. Diagn Mol Pathol 15:1952052006


Veninga HBecker SHoek RMWobus MWandel Evan der Kaa J: Analysis of CD97 expression and manipulation: antibody treatment but not gene targeting curtails granulocyte migration. J Immunol 181:657465832008


Wagner NMichiels JFSchedl AWagner KD: The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo. Oncogene 27:366236722008


Wang TWard YTian LLake RGuedez LStetler-Stevenson WG: CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells. Blood 105:283628442005


Wang ZAhmad ALi YKong DAzmi ASBanerjee S: Emerging roles of PDGF-D signaling pathway in tumor development and progression. Biochim Biophys Acta 1806:1221302010


Wang ZYQiu QQEnger KTDeuel TF: A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1. Proc Natl Acad Sci U S A 90:889689001993


Yang LHan YSuarez Saiz FMinden MD: A tumor suppressor and oncogene: the WT1 story. Leukemia 21:8688762007


Yona SLin HHSiu WOGordon SStacey M: Adhesion-GPCRs: emerging roles for novel receptors. Trends Biochem Sci 33:4915002008


Zhang LWang LRavindranathan AMiles MF: A new algorithm for analysis of oligonucleotide arrays: application to expression profiling in mouse brain regions. J Mol Biol 317:2252352002


Zhao YXiao AdiPierro CGCarpenter JEAbdel-Fattah RRedpath GT: An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9. Am J Pathol 176:303230492010


Cited By



All Time Past Year Past 30 Days
Abstract Views 54 54 29
Full Text Views 132 132 23
PDF Downloads 84 84 10
EPUB Downloads 0 0 0


Google Scholar