EGFRvIII Expression Research Articles

Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line.

Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion molecules (CD36, CD24), signal transduction related molecules (MKP-1) and other molecules related to cancer (CD98, thymosin beta-10) were altered in the EGFRvIII transfected cell line. Northern hybridisations and Western blot analyses were used to verify selected results. The results indicate that expression of EGFRvIII alters expression of genes involved in the control of cell growth, survival and motility. © 2001 Cancer Research Campaign http://www.bjcancer.com

Analysis of genomic rearrangements associated with EGRFvIII expression suggests involvement of Alu repeat elements.

We have developed a polymerase chain reaction (PCR)-based strategy for the synthesis and analysis of rearranged epidermal growth factor receptor (EGFR) fragments associated with the vIII mutant receptor expressed in glioblastomas with EGFR amplification. The sequencing of aberrant tumor fragments showed that intragenic deletion rearrangements consistently involve an approximately 600-bp region in intron 7 of EGFR and several rearrangement sites interspersed throughout the large (>100 kb) first intron of this gene. Examination of the intron 7 breakpoint region revealed an Alu repeat element, and all intron 7 rearrangement sites were located within or downstream of this repeat sequence. Analysis of intron 1 for similar sequences resulted in the identification of 11 sites containing >80% homology with parts of the Alu element in intron 7. Reverse transcriptase-PCR and/or Western analysis of the tumors showed the presence of EGFRvIII cDNAs and/or proteins, respectively, in all cases for which a rearranged genomic fragment was generated by long-range PCR. Collectively, these data suggest that EGFR rearrangements, associated with the synthesis of the most common EGFR mutant, are mediated by a specific sequence element.

Analysis of genomic rearrangements associated with EGFRvIII expression suggests involvement of Alu repeat elements

Analysis of genomic rearrangements associated with EGFRvIII expression suggests involvement of Alu repeat elements

Analysis of genomic rearrangements associated with EGFRvIII expression suggests involvement of Alu repeat elements

Analysis of genomic rearrangements associated with EGFRvIII expression suggests involvement of Alu repeat elements

Expression of activated epidermal growth factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens.

Amplification of the epidermal growth factor receptor (EGFR) is a common event in the molecular pathogenesis of high-grade astrocytic tumors, occurring in 50% of glioblastoma multiforme (GBM) cases. A subset of GBMs also express a constitutively phosphorylated truncated receptor (EGFRvIII). Expression of transfected EGFRvIII in cells has been reported to activate the Ras-mitogen-activated protein kinase pathway and to provide a growth advantage. Novel therapeutic agents targeting signal transduction pathways are entering early clinical trials; determination of which GBMs express EGFRvIII might help identify patients who might benefit from these biological agents. A cohort of 15 flash-frozen surgical specimens (12 GBMs, 2 gliosarcomas, and 1 adult low-grade glioma) were evaluated for EGFR and EGFRvIII expression and for EGFR activation status using immunohistochemical (IHC) analysis, Western blotting, and reverse transcription-polymerase chain reaction assays. Levels of activated Ras-guanosine triphosphate were measured using a nonradioactive luciferase-based technique. Mitogen-activated protein kinase activation was determined using a myelin basic protein assay. IHC analysis was performed on paraffin-embedded, formalin-fixed, pathological specimens. Normal control samples included white matter specimens distal to tumors (n = 5), a sample obtained during a lobectomy for treatment of epilepsy (n = 1), and cultured fetal human astrocytes (n = 1). We demonstrated higher levels of activated Ras and mitogen-activated protein kinase in GBM specimens, compared with normal brain tissue or the low-grade glioma. There was a very good correlation between results obtained using specialized molecular techniques and those obtained using routine IHC techniques. Screening for EGFRvIII expression may be of prognostic importance, because patients with EGFRvIII-positive tumors exhibited shorter life expectancies (mean survival time for patients with EGFRvIII-positive tumors, 4.5 +/- 0.6 mo; mean survival time for patients with EGFRvIII-negative tumors, 11.2 +/- 0.9 mo). We demonstrated that routine IHC techniques using commercially available antibodies are capable of identifying which GBM specimens express EGFRvIII and whether the EGFRs are activated. Such a molecular classification of GBMs might allow us to determine which patients might benefit from biologically targeted therapies. In addition, characterization of specimens with respect to their EGFRvIII status seems to be of prognostic value.

Differential Modulation of Mitogen-activated Protein (MAP) Kinase/Extracellular Signal-related Kinase Kinase and MAP Kinase Activities by a Mutant Epidermal Growth Factor Receptor

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.