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

Laboratory investigation

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Object

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.

Methods

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.

Results

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.

Conclusions

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: wbroaddus@mcvh-vcu.edu.

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.

Headings

Figures

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    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.

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    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.

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    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.

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    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.

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    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.

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