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Mutant Epidermal Growth Factor Receptor Up-Regulates Molecular Effectors of Tumor Invasion¹

Departments of Pathology [A. L., C. A. G., H. M. M., J. H. S, G. J. R.] and Surgery [H. S. F., G. E. A., J. H. S.], Duke University Medical Center, Durham, North Carolina 27710

	ABSTRACT

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The gene most commonly altered in human glioblastomas is the epidermalgrowth factor receptor (EGFR). We profiled transcripts induced by mutantEGFR to better understand its role in tumor progression. The pattern found suggested enhanced tumor invasion. The highly induced genes included extracellular matrix components, metalloproteases, and a serine protease. We confirmed that mutant EGFR did make glioblastoma cells both more motile and invasive using in vitro assays. Furthermore, inhibitors of EGFR (OSI-774 and Tyrphostin AG1478) selectively down-regulated these molecular effectors in glioblastoma cells, eliminating enhanced invasion.

	Introduction

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The EGFR³ is genomically amplified in 40–50% of human glioblastoma tumors (1) and often followed by gene rearrangement. The most common rearrangement is EGFRvIII, an in-frame deletion of amino acids 6–273. The resulting mutant protein is ligand-independent, constitutively phosphorylated, and localizes to the cell surface (2, 3, 4) . EGFRvIII expression enhances the tumorigenicity of glioma cells in vivo by increasing cell proliferation and decreasing cell death (5 , 6) . EGFRvIII-positive tumors have also been associated with poorer prognosis (7) and shorter life expectancies (8) . Unfortunately, little is yet known about the molecular mechanisms that EGFRvIII uses to produce a malignant phenotype.

To understand how EGFRvIII might exert its pathologic effects, we looked for downstream transcriptional targets using SAGE and other expression profiling methods. We have analyzed and compared the transcriptomes of a control and an EGFRvIII-expressing glioblastoma cell line and have identified genes of which the transcript levels were enhanced by EGFRvIII. These targets included ECM proteins, metalloproteases, and a serine protease, which point toward a role in tumor invasion. The effects of EGFR-inhibiting drugs were also evaluated in how they change gene expression. Our data suggests that not only does EGFRvIII expression enhance invasion but points to a small set of extracellular proteins eventually responsible for the malignant behavior of glioblastomas with mutant EGFR.

	Materials and Methods

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Cell Lines.
The retroviral vector, pMFG, containing either the ß-galactosidase gene or the EGFRvIII gene was used to generate replication-defective viruses from the CRIP packaging cell line. The viral supernatants were used to transfect the glioblastoma cell line, D54-MG. Stable clones expressing ß-galactosidase were designated D54-LacZ. Single cell clones expressing EGFRvIII were isolated, and a clone expressing high levels of the mutant protein was designated D54-EGFRvIII. The glioblastoma cell line U251-MG was also transfected with EGFRvIII as described above (U251-EGFRvIII) and was grown as athymic mice xenografts. The xenografts were removed, dissociated into single cells, and sorted for EGFRvIII expression using the Becton Dickinson FACsort (Franklin Lakes, NJ). These sorted U251-EGFRvIII cells were grown in vitro and compared with the parental U251 cell line. U118-MG glioblastoma cell line transfected with EGFRvIII or a vector control were the kind gift of Dr. David James (Mayo Clinic, Rochester, MN). The U87 parental glioblastoma cell line and the U87 cell line transfected with EGFRvIII have been described earlier (5) , and were grown as either cell lines or xenografts.

SAGE.
Independent SAGE libraries were constructed from the stable clones D54-lacZ and D54-EGFRvIII as described earlier (9) . Approximately 2000 plasmid clones were sequenced from each library as part of the CGAP SAGE project. SAGE tags were extracted and their tag frequencies compared using the SAGE software v 4.0. Unique transcript tags were identified as described earlier and mapped to predicted SAGE tags from primate cDNA GenBank entries or UniGene clusters that had a poly(A) signal and/or a poly(A) tail. Gene expression increases were predicted by dividing the fraction of a given SAGE tag in the EGFRvIII-expressing library by the fractional representation in the ß-galactosidase-expressing library. The complete SAGE transcript tag counts have been deposited at the National Center for Biotechnology Information SAGEmap website⁴ under the library names SAGE_Duke_H54_lacZ and SAGE_Duke_H54_EGFRvIII.

DNA Array Analysis.
Poly(A) RNA isolated from D54-lacZ and D54-EGFRvIII cell lines were used to probe the Atlas Human Cancer 1.2 cDNA Array as described by the manufacturers (Clontech Laboratories, Palo Alto, CA). Briefly, the message RNA was reverse transcribed in the presence of [-³²P]dATP to generate the probe, the membranes were prehybridized at 68°C for 30 min, hybridized overnight at 68°C, washed twice with 2x SSC/1% SDS and twice with 0.1x SSC/0.5% SDS for 30 min each at 68°C each, and then visualized by exposure to X-ray film.

Inhibitor Studies.
Stock solutions of the tyrosine kinase inhibitors Tyrphostin AG1478 (Sigma-Aldrich, St. Louis, MO) and OSI-774 (Tarceva; courtesy of Ken Iwata, OSI Pharmaceuticals, Melville, NY) were prepared in DMSO. Glioblastoma cell lines were treated with either 25 µM Tyrphostin or 20 µM OSI-774 for the indicated time points. Control cells were treated with an equal volume of DMSO. Total RNA was isolated, and cDNA was synthesized using standard techniques. Transcript levels were assayed by quantitative PCR.

Quantitative PCR.
Quantitative PCR was performed on cDNA templates using a thermocycler with continuous fluorescent monitoring capabilities (LightCycler; Roche Diagnostics) and SYBR Green I (Molecular Probes, Eugene, OR) using PCR conditions and data analysis as described earlier (10) . The integrity of the cDNA and normalization of the cDNA yields were performed using primers specific for ß-actin. Primers specific for genes induced by EGFRvIII were designed to generate 140–240-bp products, and their sequences are available on request.

Immunohistochemistry.
Immunohistochemical staining was performed on 5 µm formalin-fixed, paraffin-embedded glioblastoma tissue sections for EGFRvIII using the mouse monoclonal antibody L84A at a concentration of 3 µg/ml, for Fibrillin-1 using the mouse monoclonal clone 12A5.18 (Neomarkers, Fremont, CA) at a concentration of 3 µg/ml and for carbonic anhydrase 9 using the mouse monoclonal M75 antibody (generous gift of E. Oosterwijk, University Medical Center, Nijmegen, the Netherlands) at a dilution of 2.8 µg/ml. The slides were immersed in solvent to remove paraffin, rehydrated, and blocked for endogenous peroxidase activity. The sections were then blocked with horse serum and sequentially incubated with primary antibody (1 h, 37°C), biotinylated secondary antibody, and streptavidin-conjugated horseradish peroxidase (Super sensitive detection system; Biogenex, San Ramon, CA). Bound antibody was detected using 3,3'-diaminobenzidine and hydrogen peroxide, counterstained with 1% hematoxylin, and permanently mounted.

Scratch Assay.
D54-lacZ and D54-EGFRvIII cells were seeded in triplicates in six-well fibronectin or collagen IV coated plates (Becton Dickinson Labware, Bedford, MA). A scratch through the central axis of the plate was gently made using a pipette tip when the cells were 80% confluent. Migration of the cells into the scratch was observed at six preselected points at 0, 4, 8, 16, and 24 h, and one D54-lacZ and one D54-EGFRvIII plate were stained using Diff Quik (Dade Behring, Newark, DE) after 8, 16, and 24 h, and photographed at x10 magnification.

In Vitro Invasion Assays.
The BD Biocoat Matrigel Invasion Chamber assay was performed as described by the manufacturers (Becton Dickinson Labware). Briefly, the Matrigel inserts were rehydrated, and 2.5 x 10⁴ D54-lacZ or D54-EGFRvIII cells were added to the invasion or control chamber wells. Twenty-four h later, the cells on the upper side of the chamber were scraped, and the ones on the lower side of the chamber were stained with Diff Quik (Dade Behring) and counted using light microscopy with a standardized grid. The cells were treated with either 0, 10, or 20 µM OSI-774 for 24 h prior and while in the invasion chambers, for a total inhibitor exposure time of 48 h. All of the experimental and control groups were done in triplicates. The percentage of invasion was calculated by dividing the mean number of cells invading through the Matrigel with those that migrate through the control. The invasion index was calculated by taking the ratio of the percentage of invasion in D54-EGFRvIII with that in D54-lacZ.

	Results and Discussion

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Model System.
Our experimental model was to constitutively overexpress EGFRvIII in glioblastoma cells using retroviral-mediated gene transfer. Stable cell populations expressing either the ß-galactosidase gene (D54-lacZ) or the EGFR deletion mutant, EGFRvIII (D54-EGFRvIII), were generated in the human glioblastoma (GBM) cell line, D54-MG. EGFRvIIItranscript levels were 42-fold higher in D54-EGFRvIII when compared with D54-lacZ using real-time PCR (data not shown). D54-MG was chosen because it had one of the lowest EGFRtranscript levels for a cultured glioblastoma cell line (11) . Flow cytometry using an EGFRvIII-specific antibody revealed that the mutant protein was expressed and detected on the cell surface of D54-EGFRvIII (data not shown).

Expression Analysis.
To find downstream targets of EGFRvIII, SAGE libraries were constructed from D54-EGFRvIII and D54-lacZ. A total of 124,177 SAGE tags representing 22,945 unique transcript tags derived from the two SAGE libraries were compared. There were 70 transcripts with 5-fold different expression below a 0.01 P chance. Of these 70 transcripts, 38 were induced by the stable expression of EGFRvIII and the remainder repressed. The complete transcript counts can be viewed or downloaded from the SAGEmap website. We concentrated our efforts on a subset of induced genes shown in Table 1 , which included ECM proteins, some metalloproteases, a collagen, and a serine protease. Using a DNA filter array, MMP13 was additionally identified as up-regulated by EGFRvIII (data not shown). The transcript tag levels for this gene were too low to yield a significant difference for the amount of sequencing performed and, therefore, missed by SAGE alone.

A comparison of the expression levels of the EGFRvIII target genes in normal tissues using the public gene expression database, SAGEmap, revealed that most of these genes were either absent or expressed at very low levels in most normal tissues (Fig. 1) . The exception was cultured fetal astrocytes. However, these astrocytes were cultured with supplemental epidermal growth factor in the medium, perhaps inducing some of these genes. The specific induction of the genes listed in Table 1 using mutant EGFR protein in cultured cells, in conjunction with a lack of expression in normal adult brain, suggested that additional investigation was warranted.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Virtual Northern of three EGFRvIII target genes. SAGE libraries from a variety of normal and cancer tissues deposited in the public gene expression database SAGEmap were used to display expression levels of the EGFRvIII-induced genes, Collagen VIII A1 (COL8A1, alternative transcript), FAP, and Fibrillin-1 (FBN1). The SAGE libraries shown are the GBM cell lines expressing either EGFRvIII (D54-EGFRvIII) or ß-galactosidase (D54-LacZ) and various normal tissues. Fetal astrocytes are untransformed human astrocytes grown in a short-term cell culture. Blank ovals indicate no detectable expression. Progressive shading indicates increasing levels of expression to a maximum of 128/100,000 SAGE tags for the black oval.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. EGFRvIII transcriptional targets are inhibited by the EGFR-targeting drugs Tyrphostin AG1478 and OSI-774. A, in a short time course, D54-EGFRvIII cells were treated with DMSO solvent or 25 µM Tyrphostin AG1478 for 6 h (light gray columns), 12 h (dark gray columns), or 24 h (black columns). Transcript levels were analyzed by quantitative PCR and expressed as a percentage of the total transcript levels in the control (DMSO-treated) cells. COL8A1' refers to a longer alternative transcript of COL8A1. B, OSI-774 reproducibly inhibited EGFRvIII targets at 48 h. D54-EGFRvIII cells were treated with DMSO or 20 µM OSI-774, and the transcript levels of the EGFRvIII targets were analyzed by quantitative PCR. Relative transcript levels of the EGFRvIII-regulated genes in the controls (black columns) and the OSI-774-treated cells (gray columns) are plotted. Transcript levels of HOG18, HXB, and IGFBP3hypoxia-regulated genes not influenced by EGFRvIII were used as controls and were unaffected by Tyrphostin AG1478 (A) and OSI-774 (B); bars, ±SD.

In Vivo Expression.
The next question was if any of these genes were induced in human tumors in addition to our cell culture models. Primary glioblastoma tissue sections were stained with an EGFRvIII-specific antibody that allowed us to classify GBMs as either EGFRvIII-positive or EGFRvIII-negative (Fig. 3) . Two cases of each type were immunostained for Fibrillin-1, a component of the ECM. The two EGFRvIII-positive tumors were strongly positive for Fibrillin-1 (Fig. 3, B and E) . In the case of one of them, strong staining around vessel structures was seen (Fig. 3B) in addition to other regions of the tumor. The EGFRvIII-negative tumors did not express Fibrillin-1 (Fig. 3, H and K) . However, restricted staining around some vessel structures was observed in one of them (Fig. 3K) . To rule out any differences in the tissue preparation that might produce the staining observed, control staining was performed with carbonic anhydrase IX antibody, a hypoxia marker (10) , and mouse IgG. There was an independent pattern of hypoxia staining in all four of the sections (Fig. 3, C, F, I, and L) , and staining with mouse IgG was negative (data not shown). In summary, Fibrillin-1 and EGFRvIII protein levels colocalized to the same region in all five of the glioblastomas tested using immunohistochemistry.

View larger version (198K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. In vivo expression of Fibrillin-1 in primary glioblastoma tumors. IHC with a monoclonal antibody specific for EGFRvIII was used to classify primary glioblastomas as EGFRvIII-positive (A and D) or EGFRvIII-negative (G and J). In vivo expression of Fibrillin-1 by IHC (B, E, H, and K) correlated with EGFRvIII IHC (first column). Sections were stained with carbonic anhydrase 9 (C, F, I, and L), a hypoxia marker, as a control showing an independent pattern of staining. Original magnification is x10 for all panels.

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Comparison of the in vitro migration and invasion abilities of D54-lacZ and D54-EGFRvIII. A, EGFRvIII-expressing cells show higher motility in a standard scratch assay. The migration of D54-lacZ and D54-EGFRvIII cells was qualitatively assessed 8 h and 16 h after the introduction of a scratch in monolayer cultures of these cells grown on fibronectin. The gray dotted lines represent the position of the initial scratch. B, the number of D54-EGFRvIII cells that invade through the Matrigel decreases as the concentration of OSI-774 increases (solid line), but the D54-lacZ (dotted line) cells show no effect or a slight increase. The Y-axis represents the number of cells invading per mm2; bars, ±SD.

Genes Promoting Invasion.
EGFR and, more recently, EGFRvIII have been implicated in invasion and a more malignant-acting tumor. In ovarian cancer, antisense EGFR and EGFR inhibitors repressed the invasive phenotype (16) . Also, in small cell lung cancer, EGFRvIII enhanced in vitro invasion to levels comparable with those found in our study on GBMs (17) . It is known that EGFRvIII enhances the tumorigenicity of GBMs. Insight is provided into the molecular mechanism of this action by the EGFRvIII-induced genes (Table 1) .

Tumor cell invasion is a complex and multistep process involving interaction of the tumor cell with the extracellular barrier, proteolytic digestion of this ECM, and, finally, cell migration through the space created. MMPs aid in invasion by degrading a broad range of ECM components in gliomas and in other tumors. Both MMP1 and MMP13 are collagenases that have been implicated in tumor cell invasion. MMP13 is primarily expressed by malignant tumor cells and has been shown to markedly increase the invasion of fibrosarcoma cells through type I collagen and Matrigel (18) . MMP1 expression has been related to the invasive property of a variety of cancers including melanoma (19) and ovarian tumors (20) . FAP , also known as Seprase, is an integral membrane serine protease that has been associated with the invasive behavior of melanoma (21) and breast carcinoma cells (22) . ECM components like COL8A1 and Fibrillin-1 were also up-regulated in our model. Fibrillin-1 protein expression was correlated in this study with EGFRvIII in human glioblastomas. Our study suggests that the accumulation of a number of the above protein products assist in glioblastoma invasion.

In addition to genes with a potential role in tumor invasion, IL13RA1 was interesting because it had been identified previously as a marker for GBM (23) . IL13R expressed in GBM tissue is both quantitatively and qualitatively different from IL13R expressed in normal tissues, and is being exploited currently for therapeutic purposes (23) . Our studies indicate that expression of IL13R is at least partially influenced by EGFRvIII. Additional downstream targets of EGFRvIII might be exploited as therapeutic targets or as biomarkers for the pathologic effects of the mutant gene.

In conclusion, EGFRvIII induces a specific pattern of genes, and many of these genes are likely effectors of tumor invasion. The two drugs tested that target EGFR both selectively inhibit the expression of the invasive genes. The targets of EGFRvIII identified by our study provide insight into the molecular mechanism of EGFRvIII-enhanced invasion and are potential tumor markers or biomarkers for drug screens of EGFR inhibition.

	ACKNOWLEDGMENTS

 
We thank Robert L. Strausberg (NCI, Bethesda, MD) for CGAP planning and administration, Christa Prange (Lawrence Livermore National Laboratory, Livermore, CA) for CGAP library arraying, Jeff Touchman and Gerard Bouffard (NIH Intramural Sequencing Center, Bethesda, MD) for CGAP sequencing and informatics, Alex Lash (National Center for Biotechnology Information, Bethesda, MD) for SAGEmap informatics, Glenn Dranoff (Dana-Farber Cancer Institute, Boston, MA) for the EGFRvIII retrovirus, Roger McLendon (Duke University) for expert histopathology, Darell Bigner (Duke University) for EGFRvIII antibody, Ashani Weeraratna (National Human Genome Research Institute, Bethesda, MD) for invasion assay expertise, and technical assistance from Elizabeth M. Moody and Linda Cleveland (Duke University). We also thank I-Mei Siu, Robert Beaty, Deric Olschner, and Brian Fee (Duke University) for the construction of CGAP SAGE libraries from normal tissues.

	FOOTNOTES

 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the CGAP (NCI contract S98–146), the W. M. Keck Foundation, and NCI Grant U01 CA88128.

² To whom requests for reprints should be addressed, at Duke University Medical Center, Box 3156, Durham, NC 27710. Phone: (919) 684-5343; Fax: (919) 681-2796; E-mail: greg.riggins{at}duke.edu

³ The abbreviations used are: EGFR, epidermal growth factor receptor; ECM, extracellular matrix; SAGE, Serial Analysis of Gene Expression; GBM, glioblastoma multiforme; CGAP, Cancer Genome Anatomy Project; poly(A), polyadenylic acid; MMP, matrix metalloproteinase; FAP, fibroblast activation protein; IHC, immunohistochemistry.

⁴ Internet address: http://www.ncbi.nlm.nih.gov/SAGE/.

Received 2/ 7/02. Accepted 5/ 1/02.

	REFERENCES

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