EGFR and erbB2 in malignant peripheral nerve sheath tumors and implications for targeted therapy

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas with poor prognosis and limited treatment options. Evidence for a role of epidermal growth factor receptor (EGFR) and receptor tyrosine kinase erbB2 in MPNSTs led us to systematically study these potential therapeutic targets in a larger tumor panel (n = 37). Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization analysis revealed increased EGFR dosage in 28% of MPNSTs. ERBB2 and three tumor suppressor genes (PTEN [phosphatase and tensin homolog deleted on chromosome 10], CDKN2A [cyclin-dependent kinase inhibitor 2A], and TP53 [tumor protein p53]) were frequently lost or reduced. Reduction of CDKN2A was linked to appearance of metastasis. Comparison of corresponding neurofibromas and MPNSTs revealed an increase in genetic lesions in MPNSTs. No somatic mutations were found within tyrosine-kinase-encoding exons of EGFR and ERBB2. However, at the protein level, expression of EGFR and erbB2 was frequently detected in MPNSTs. EGFR expression was significantly associated with increased EGFR gene dosage. The EGFR ligands transforming growth factor alpha and EGF were more strongly expressed in MPNSTs than in neurofibromas. The effects of the drugs erlotinib and trastuzumab, which target EGFR and erbB2, were determined on MPNST cell lines. In contrast to trastuzumab, erlotinib mediated dose-dependent inhibition of cell proliferation. EGF-induced EGFR phosphorylation was attenuated by erlotinib. Summarized, our data indicate that EGFR and erbB2 are potential targets in treatment of MPNST patients.

Fig. 1.

Genetic alterations, receptor expression, and morphology of malignant peripheral nerve sheath tumors (MPNSTs) and MPNST cell cultures. (A) Electropherograms depicting gene dosage of MPNST 21914 (lower panel) and corresponding plexiform neurofibroma (pNF) 21912 (upper panel) generated with the SALSA P105 Oligodendroglioma-2 multiplex ligation-dependent probe amplification kit (Vs. 04). The arrows indicate signal reduction of the five CDKN2A probes in the MPNSTs. The star marks the increased signal of the EGFR exon 1 probe (inset). Fluorescence in situ hybridization analysis demonstrates cluster amplification of epidermal growth factor receptor gene EGFR (red signals; centromer: green signals). (B) Immunohistochemistry of EGFR on MPNST 24324. Note the strong EGFR expression in the cellular MPNSTs compared to the area with differentiation corresponding to pNF. Original magnification: ×200; right corner, ×400. (C) Receptor tyrosine-protein kinase erbB2 immunofluorescence of MPNST 24626. Original magnification: ×400. (D and E) Morphology of low-passage MPNST culture 31002 and MPNST cell line NSF-1, respectively.

Fig. 2.

Detection of epidermal growth factor receptor (EGFR), EGFR ligands, and receptor tyrosine-protein kinase (erbB2). (A) Western blot showing EGFR and erbB2 expression in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cell lines, fibroblasts (Fibrobl.), and neurofibromas (NF). Lysate of EGF-stimulated A431 carcinoma cells served as EGFR positive control. Detection of β-actin demonstrates equal loading. (B) Different EGFR isoforms in MPNSTs and plexiform neurofibromas (pNFs) separated on the same gel. (C) Detection of phosphorylated proteins using the p-Tyr antibody specific for phosphotyrosine residues. Two sets of A431 and 21914 lysates were run on the same gel and blotted. After cutting the membrane one set was incubated with an EGFR antibody, the other set was incubated with p-Tyr. MM, MagicMark XP size standard. (D) Expression of EGFR ligands EGF and transforming growth factor α (TGFA) was determined by reverse transcriptase PCR in neurofibromas, MPNSTs, and MPNST cell lines ST88-14 and S462. RPS3 served as housekeeping gene. Abbreviations: M, size standard PUC 19 DNA/MspI; GBM, glioblastoma cell line DBTRG-05 MG, serving as positive control; dNF, dermal neurofibromas.

Fig. 3.

Effect of erlotinib and imatinib on proliferation and epidermal growth factor receptor (EGFR) phosphorylation. (A) Effect of different erlotinib concentrations on cell proliferation in four malignant peripheral nerve sheath tumor lines after 7 days of incubation. (B) Single and combination (Combi) treatment with 5 μM of erlotinib and imatinib. Error bars represent the SEM of three independent experiments. ST88-14 in B was tested once in duplicate. (C) Effect of erlotinib on EGF-induced EGFR phosphorylation. The cells were cultivated 24 h without serum and then treated with vehicle, EGF (100 ng/ml), or erlotinib (5 μM) plus EGF. (D) Cells were stimulated with 50 ng/ml EGF. The phosphorylated form of EGFR (pEGFR) was detected by a phosphospecific EGFR antibody. Rehybridization of the membranes with an EGFR antibody shows equal loading.