Activation of multiple ERBB family receptors mediates glioblastoma cancer stem-like cell resistance to EGFR-targeted inhibition

Abstract

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.

Figure 1

Characterization of GBM CSCs. Sphere-forming GBM CSCs from surgical specimens were obtained after 2 to 4 weeks of culture in stem cell medium (A) that expressed NSC markers (B: CD133, green; DAPI nuclear counterstain, blue; C: nestin, green) and demonstrated multilineage potential (D: βIII-tubulin/Tuj1, green; GFAP, red). Enrichment for CD133 was verified using flow cytometry (G). GBM CSCs initiated tumors 12 to 16 weeks after orthotopic injection, as evidenced by MRI (E: 22 GBM CSCs, 100,000 cells, 106 days after injection; T1-weighted with gadolinium enhancement) and histology (F: hematoxylin and eosin). T indicates tumor.

Figure 2

EGFR signaling components are present in GBM CSCs. Immunocytochemistry demonstrated expression of EGFR on both NSCs (A) and GBM CSCs (B). EGFR expression was validated for all GBM CSC lines using Western immunoblot analysis (C), semiquantitative PCR (D), and flow cytometry (E and F). Flow cytometry analysis showed presence of EGFR expression in all GBM CSC lines, although at varying levels, and NSCs demonstrated a significantly higher percentage of EGFR⁺ cells compared to GBM CSCs (F). Presence of the common EGFRvIII mutation was tested using semiquantitative RT-PCR and was not present in any of the isolated GBM CSC lines (D).

Figure 3

GBM CSCs propagate independent of exogenous EGF with increased activation of ERBB family receptors. Removal of exogenous EGF and bFGF significantly reduced short-term (4 days) proliferation of NSCs and GBM CSCs (*P < .05), although the antiproliferative effect was less in the GBM CSC lines compared to NSCs (A). Unlike NSCs that growth arrested without addition of exogenous EGF and bFGF, GBM CSCs continued to propagate in vitro as spheres (B). GBM CSCs without exogenousmitogens maintained expression of the stem cell marker nestin (C: green; DAPI nuclear counterstain, blue),multilineage potential (D: βIII-tubulin/Tuj1, green; GFAP, red), and tumor initiation capacity (E: 22 GBM CSC -EGF/-bFGF, 100,000 cells, 90 days). Flow cytometry analysis of GBM CSC -EGF/-bFGF demonstrated significantly decreased CD133 expression and increased EGFR expression (G: *P < .05). CD133 expression did not return on mitogen readdition and subculture, whereas EGFR expression decreased (G). Immunoblot analysis demonstrated increased ERBB family activation (ERBB2, ERBB3) and maintained downstream pAKT and pERK1/2 in GBM CSCs without EGF and bFGF compared to +EGF/+bFGF (F).

Figure 4

Dual blockade of EGFR and ERBB2 prevents propagation of GBM CSCs. Cetuximab (inhibitory EGFR antibody, 100 nM) inhibited colony-forming ability of GBM CSCs, but the effect is heavily attenuated in GBM CSCs without exogenous EGF and bFGF (A, B) (*P < .05). Lapatinib (EGFR/ERBB2 TKI, 5 µM) inhibited colony formation by GBM CSCs from either growth condition equally (A, B). Lapatinib also significantly better prevented colony formation in an additional four GBM CSC lines, compared to cetuximab (C). Immunoblot analysis demonstrated reduced AKT activation with lapatinib after EGF stimulation, as well as reduced pERK1/2 in three of the GBM CSC lines (22, 12.1, and 44) (D).