BACKGROUND: Amplification of the epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are among the most common genetic alterations in glioblastoma (GBM), the most frequent and most aggressive primary brain tumor. METHODS: In the present work, we analyzed the clonal evolution of these major EGFR aberrations in a small cohort of GBM patients using a unique surgical multisampling technique. Furthermore, we overexpressed both receptors separately and together in 2 patient-derived GBM stem cell lines (GSCs) to analyze their functions in vivo in orthotopic xenograft models. RESULTS: In human GBM biopsies, we identified EGFR amplification as an early event because EGFRvIII mutations emerge from intratumoral heterogeneity later in tumor development. To investigate the biological relevance of this distinct developmental pattern, we established experimental model systems. In these models, EGFR+ tumor cells showed activation of classical downstream signaling pathways upon EGF stimulation and displayed enhanced invasive growth without evidence of angiogenesis in vivo. In contrast, EGFRvIII+ tumors were driven by activation of the prototypical Src family kinase c-Src that promoted VEGF secretion leading to angiogenic tumor growth. CONCLUSIONS: The presented work shows that sequential EGFR amplification and EGFRvIII mutations might represent concerted evolutionary events that drive the aggressive nature of GBM by promoting invasion and angiogenesis via distinct signaling pathways. In particular, c-SRC may be an attractive therapeutic target for tumors harboring EGFRvIII as we identified this protein specifically mediating angiogenic tumor growth downstream of EGFRvIII.

Department of Biomedicine University of Bergen Norway

Neuro Oncol. 2016 Dec;18(12):e2. doi: 10.1093/neuonc/now271. PubMed

Zobrazit více v PubMed

Fuller GN, Bigner SH. Amplified cellular oncogenes in neoplasms of the human central nervous system. Mutat Res. 1992;276(3):299–306. PubMed

Wong AJ, Ruppert JM, Bigner SH et al. . Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci USA. 1992;89(7):2965–2969. PubMed PMC

Wei Q, Clarke L, Scheidenhelm DK et al. . High-grade glioma formation results from postnatal pten loss or mutant epidermal growth factor receptor expression in a transgenic mouse glioma model. Cancer Res. 2006;66(15):7429–7437. PubMed

Li L, Dutra A, Pak E et al. . EGFRvIII expression and PTEN loss synergistically induce chromosomal instability and glial tumors. Neuro Oncol. 2009;11(1):9–21. PubMed PMC

Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell plasticity. Nature. 2013;501(7467):328–337. PubMed PMC

Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature. 2013;501(7467):355–364. PubMed PMC

Rosland GV, Engelsen AS. Novel points of attack for targeted cancer therapy.Basic Clin Pharmacol Toxicol. 2014;116(1):9–18. PubMed PMC

Sottoriva A, Spiteri I, Piccirillo SG et al. . Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci USA. 2013;110(10):4009–4014. PubMed PMC

Patel AP, Tirosh I, Trombetta JJ et al. . Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014;344(6190):1396–1401. PubMed PMC

Francis JM, Zhang CZ, Maire CL et al. . EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing. Cancer Discov. 2014;4(8):956–971. PubMed PMC

Pedersen MW, Tkach V, Pedersen N, Berezin V, Poulsen HS. Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility. Int J Cancer. 2004;108(5):643–653. PubMed

Maity A, Pore N, Lee J, Solomon D, O'Rourke DM. Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3’-kinase and distinct from that induced by hypoxia. Cancer Res. 2000;60(20):5879–5886. PubMed

Magnus N, Garnier D, Rak J. Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells. Blood. 2010;116(5):815–818. PubMed

Martens T, Laabs Y, Gunther HS et al. . Inhibition of glioblastoma growth in a highly invasive nude mouse model can be achieved by targeting epidermal growth factor receptor but not vascular endothelial growth factor receptor-2. Clin Cancer Res. 2008;14(17):5447–5458. PubMed

Guillamo JS, de Bouard S, Valable S et al. . Molecular mechanisms underlying effects of epidermal growth factor receptor inhibition on invasion, proliferation, and angiogenesis in experimental glioma. Clin Cancer Res. 2009;15(11):3697–3704. PubMed

Bonavia R, Inda MM, Vandenberg S et al. . EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway. Oncogene. 2012;31(36):4054–4066. PubMed PMC

Clarke K, Smith K, Gullick WJ, Harris AL. Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway. Br J Cancer. 2001;84(10):1322–1329. PubMed PMC

Jin X, Yin J, Kim SH et al. . EGFR-AKT-Smad signaling promotes formation of glioma stem-like cells and tumor angiogenesis by ID3-driven cytokine induction. Cancer Res. 2011;71(22):7125–7134. PubMed

Feng H, Hu B, Vuori K et al. . EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180. Oncogene. 2013;33(19):2504–2512. PubMed PMC

Katanasaka Y, Kodera Y, Kitamura Y, Morimoto T, Tamura T, Koizumi F. Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma. Mol Cancer. 2013;12:31. PubMed PMC

Boockvar JA, Kapitonov D, Kapoor G et al. . Constitutive EGFR signaling confers a motile phenotype to neural stem cells. Mol Cell Neurosci. 2003;24(4):1116–1130. PubMed

Nathanson DA, Gini B, Mottahedeh J et al. . Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA. Science. 2014;343(6166):72–76. PubMed PMC

Sakariassen PO, Prestegarden L, Wang J et al. . Angiogenesis-independent tumor growth mediated by stem-like cancer cells. Proc Natl Acad Sci USA. 2006;103(44):16466–16471. PubMed PMC

Campos B, Wan F, Farhadi M et al. . Differentiation therapy exerts antitumor effects on stem-like glioma cells. Clin Cancer Res. 2010;16(10):2715–2728. PubMed

Podergajs N, Brekka N, Radlwimmer B et al. . Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF. Radiol Oncol. 2013;47(4):330–337. PubMed PMC

Talasila KM, Soentgerath A, Euskirchen P et al. . EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis. Acta Neuropathol. 2013;125(5):683–698. PubMed PMC

Foerster S, Kacprowski T, Dhople VM et al. . Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics. Proteomics. 2013;13(21):3131–3144. PubMed

Huszthy PC, Giroglou T, Tsinkalovsky O et al. . Remission of invasive, cancer stem-like glioblastoma xenografts using lentiviral vector-mediated suicide gene therapy. PLoS One. 2009;4(7):e6314. PubMed PMC

Keunen O, Johansson M, Oudin A et al. . Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci USA. 2011;108(9):3749–3754. PubMed PMC

Taxt T, Jirik R, Rygh CB et al. . Single-channel blind estimation of arterial input function and tissue impulse response in DCE-MRI.IEEE Trans Biomed Eng. 2012;59(4):1012–1021. PubMed

Sottoriva A, Spiteri I, Shibata D, Curtis C, Tavare S. Single-molecule genomic data delineate patient-specific tumor profiles and cancer stem cell organization. Cancer Res. 2013;73(1):41–49. PubMed PMC

Piccirillo SG, Spiteri I, Sottoriva A et al. . Contributions to drug resistance in glioblastoma derived from malignant cells in the sub-ependymal zone. Cancer Res. 2014;75(1):194–202. PubMed PMC

Ji H, Wang J, Nika H et al. . EGF-induced ERK activation promotes CK2-mediated disassociation of alpha-Catenin from beta-Catenin and transactivation of beta-Catenin. Mol Cell. 2009;36(4):547–559. PubMed PMC

Glading A, Chang P, Lauffenburger DA, Wells A. Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway. J Biol Chem. 2000;275(4):2390–2398. PubMed

Lu KV, Zhu S, Cvrljevic A et al. . Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients. Cancer Res. 2009;69(17):6889–6898. PubMed PMC

Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Ann Rev Cell Dev Bio. 1997;13:513–609. PubMed

Angers-Loustau A, Hering R, Werbowetski TE, Kaplan DR, Del Maestro RF. SRC regulates actin dynamics and invasion of malignant glial cells in three dimensions.Mol Cancer Res. 2004;2(11):595–605. PubMed

Nishikawa R, Ji XD, Harmon RC et al. . A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA. 1994;91(16):7727–7731. PubMed PMC

Nagane M, Coufal F, Lin H, Bogler O, Cavenee WK, Huang HJ. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res. 1996;56(21):5079–5086. PubMed

Tykocinski ES, Grant RA, Kapoor GS et al. . Use of magnetic perfusion-weighted imaging to determine epidermal growth factor receptor variant III expression in glioblastoma. Neuro Oncol. 2012;14(5):613–623. PubMed PMC

Cvrljevic AN, Akhavan D, Wu M et al. . Activation of Src induces mitochondrial localisation of de2–7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism. J Cell Sci. 2011;124(Pt 17):2938–2950. PubMed PMC

Wiener JR, Nakano K, Kruzelock RP, Bucana CD, Bast RC Jr, Gallick GE. Decreased Src tyrosine kinase activity inhibits malignant human ovarian cancer tumor growth in a nude mouse model. Clin Cancer Res. 1999;5(8):2164–2170. PubMed

Fleming RY, Ellis LM, Parikh NU, Liu W, Staley CA, Gallick GE. Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by activity of src kinase. Surgery. 1997;122(2):501–507. PubMed

Mukhopadhyay D, Tsiokas L, Zhou X-M, Foster D, Brugge JS, Sukhatme VP. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 1995;375(6532):577–581. PubMed

Huveldt D, Lewis-Tuffin LJ, Carlson BL et al. . Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion. PLoS One. 2013;8(2):e56505. PubMed PMC