[CANCER RESEARCH 62, 3335–3339, June 15, 2002] Advances in Brief Mutant Epidermal Growth Factor Receptor Up-Regulates Molecular Effectors of Tumor Invasion 1 Anita Lal, Chad A. Glazer, Holly M. Martinson, Henry S. Friedman, Gary E. Archer, John H. Sampson, and Gregory J. Riggins 2 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 The gene most commonly altered in human glioblastomas is the epi- dermal growth factor receptor (EGFR). We profiled transcripts induced by mutant EGFR 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 en- hanced invasion. Introduction The EGFR 3 is genomically amplified in 40 –50% of human glio- blastoma 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-indepen- dent, constitutively phosphorylated, and localizes to the cell surface (2– 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, met- alloproteases, 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 ma- lignant behavior of glioblastomas with mutant EGFR. Materials and Methods Cell Lines. The retroviral vector, pMFG, containing either the -galacto- sidase 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 - galactosidase were designated D54-LacZ. Single cell clones expressing EGFR- vIII 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 Dick- inson 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 -galactosidase-expressing library. The complete SAGE transcript tag counts have been deposited at the National Center for Bio- technology Information SAGEmap website 4 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 [- 32 P]dATP to generate the probe, the membranes were prehybridized at 68°C for 30 min, hybridized overnight at 68°C, washed twice with 2 SSC/1% SDS and twice with 0.1 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 Tyr- phostin 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 M Tyrphostin or 20 M 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 -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 m formalin-fixed, paraffin-embedded glioblastoma tissue sections for EGFRvIII using the mouse monoclonal antibody L84A at a concentration of 3 Received 2/7/02; accepted 5/1/02. 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@duke.edu. 3 The abbreviations used are: EGFR, epidermal growth factor receptor; ECM, extra- cellular matrix; SAGE, Serial Analysis of Gene Expression; GBM, glioblastoma multi- forme; 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/. 3335 Research. on March 24, 2016. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from