Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis

Abstract

Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFR(vIII) do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.

Fig. 1.

Localized somatic expression of EGFR in adult mouse brains form tumors. (A) Schematic representation of the activation of the conditional EGFR transgenes. A strong ubiquitous promoter (CAGGS) is positioned upstream of a transcriptional stop cassette (STOP) which is flanked by 2 loxP sites (blue triangles), followed by either wild-type (WT) or mutated (vIII) human EGFR cDNAs and a polyA signal sequence (purple rectangle). The expression of EGFR is conditional to the removal of the stop cassette, which is mediated by an adenovirus expressing Cre recombinase. (B) Tumor-free survival (Kaplan-Meier) analysis of Ad-Cre-injected conditional EGFR cohorts of mice with the indicated genotypes. EGFR^WT/WT, n = 6; EGFR^vIII/vIII, n = 5. For EGFR^WT/WT, EGFR^vIII/vIII, and EGFR^WT/vIII, all on an InkΔ2/3^−/− and PTEN^−/− background, n = 33, n = 4, and n = 8, respectively.

Fig. 2.

Neuropathological analysis of EGFR tumors. Representative photomicrographs of Col1α1-EGFR; InkΔ2/3^−/−; PTEN^−/− tumor sections stained with H&E. Expression of both WT and vIII EGFR result in similar tumor phenotypes. (A) Tumors are set on a fibrilary background and contain pleimorphic nuclei (white arrow) and mitoses (black arrow) (Scale bar: 15 μM.) (B) Tumors show marked pseudopallisading necrosis. (Scale bar: 62.5 μM.) (C) Tumor cells tend to accumulate around neurons, a common feature known as perineuronal satellitosis. The highly infiltrative nature of EGFR tumor cells is depicted. (Scale bar: 30 μM.) (D) Tumor cells (black arrowheads) are infiltrating white matter tracts such as the corpus callosum (CC) and the perivascular space (white arrowhead) and (E) migrate within the subarachnoid space. (Scale bars: 30 μM and 62.5 μM, respectively.) (F) IHC stain for human EGFR showing tumor cells infiltrating normal brain. (Scale bar: 62.5 μM.)

Fig. 3.

EGFR GBM tumors express markers of astrocytic differentiation. Representative photomicrographs of GBM tumors of the indicated genotypes stained with H&E, for expression of EGFR, the astrocytic markers glial fibrillary acidic protein (GFAP) and S100 and the neuronal marker NeuN by IHC. N, normal brain; T, tumor. (Scale bars: 62.5 μM.)

Fig. 4.

Signaling networks usurped by EGFR in GBM tumor cells. STAT3 and MEK/ERK are activated by EGFR^WT but not EGFR^vIII receptors. Ex vivo cultures of cells from the indicated tumor genotypes were starved for 24 hr and stimulated with 50 ng/ml of EGF for 5 min. Immunoblots of total cell lysates were probed with the indicated antibodies.

Fig. 5.

EGFR^vIII signals differently than EGFR^WT. EGF ligand stimulation of EGFR^WT-expressing cells stimulates the activation of mTORC1, whereas mTORC2 appears to be constitutively activated in EGFR^vIII-expressing GBM tumor cells. Western blot analysis of total cell extracts from GBM tumor cells expressing the indicated receptors. Cells were serum starved and stimulated as described previously. Immunoblots were probed with antibodies as indicated.