Mutant EGFR is required for maintenance of glioma growth in vivo, and its ablation leads to escape from receptor dependence

Abstract

Epidermal growth factor receptor (EGFR) gene amplification is the most common genetic alteration in high-grade glioma, and approximately 50% of EGFR-amplified tumors also harbor a constitutively active mutant form of the receptor, DeltaEGFR. Although DeltaEGFR greatly enhances tumor growth and is thus an attractive target for anti-glioma therapies, recent clinical experiences with EGFR kinase inhibitors have been disappointing, because resistance is common and tumors eventually recur. Interestingly, it has not been established whether DeltaEGFR is required for maintenance of glioma growth in vivo, and, by extension, if it truly represents a rational therapeutic target. Here, we demonstrate that in vivo silencing of regulatable DeltaEGFR with doxycycline attenuates glioma growth and, therefore, that it is crucial for maintenance of enhanced tumorigenicity. Similar to the clinical experience, tumors eventually regained aggressive growth after a period of stasis, but interestingly, without re-expression of DeltaEGFR. To determine how tumors acquired this ability, we found that a unique gene, KLHDC8, herein referred to as SDeltaE (Substitute for DeltaEGFR Expression)-1, is highly expressed in these tumors, which have escaped dependence on DeltaEGFR. SDeltaE-1 is also expressed in human gliomas and knockdown of its expression in DeltaEGFR-independent "escaper" tumors suppressed tumor growth. Taken together, we conclude that DeltaEGFR is required for both glioma establishment and maintenance, and that gliomas undergo selective pressure in vivo to employ alternative compensatory pathways to maintain aggressiveness in the event of EGFR silencing. Such alternative pathways function as substitutes for DeltaEGFR signaling and should therefore be considered as potential targets for additional therapy.

Fig. 1.

 ΔEGFR-dependent tumor growth of U373MG cells expressing doxycycline-regulatable ΔEGFR. (A) Western blot analysis of proteins from whole-cell lysates with the indicated antibodies of engineered U373MG cells cultured in medium containing 1 μg/mL dox for 0, 1, 2, 3, and 4 days. (B) Tumor growth of U373MG cells (3 × 10⁶) expressing dox-regulatable ΔEGFR injected s.c. into the flanks of nude mice that received dox-containing drinking water as indicated by arrows at 0, 32, or 48 days. One group of mice received U373MG cells expressing kinase-defective ΔEGFR (DK) and no doxycycline. Similar experiments were done at least twice with consistent results. (mean ± SEM) (C) Western blot analysis of lysates from mouse tumors in A that received no dox or received dox at day 32 or 48.

Fig. 2.

Growth characteristics and pathway profiling of U373MG tumors upon silencing of ΔEGFR expression. Mice bearing established s.c. U373MG dox-regulatable ΔEGFR tumors remained on normal drinking water [Dox (−)], or were administered dox-containing drinking water for 4 or 8 days [Dox (+)], and tumors were resected and analyzed. (A) Profileration index as measured by counting proportion of K _i-67-positive cells (mean ± SD; *, P < 0.001, Student’s t test). ( B) Apoptotic index as measured by percentage of TUNEL-positive cells (mean ± SD; *, P < 0.05, Student’s t test). (C) Western blot analysis of proteins from tumor lysates with the indicated antibodies. Each lane represents a tumor from an individual mouse.

Fig. 3.

Relapse of U373MG tumors after silencing of ΔEGFR expression. (A) Tumor growth of U373MG cells (3 × 10⁶) expressing dox-regulatable ΔEGFR injected s.c. into the flanks of nude mice administered dox-containing drinking water 32 days after injection. Each line represents an individual growth curve of a tumor in a mouse from one typical experiment. Horizontal arrows indicate representative cases of ΔEGFR-independent “escaper” tumors. (B) Proliferation index as measured by counting proportion of K _i-67 positive cells in escaper and ΔEGFR-dependent (with Dox for 8 days from day 32 or without Dox) tumors (mean ± SD; P < 0.0001, ANOVA; *, P < 0.05; **, P < 0.01, Tukey–Kramer HSD test). (C) Apoptotic index in same tumors as part (B), as measured by TUNEL staining (mean ± SD; P < 0.0001, ANOVA; **, P < 0.01, Tukey–Kramer HSD test).

Fig. 4.

Analysis of ΔEGFR-dependent vs. escaper tumor protein expression and signaling pathway preferences. Western blot analysis with the indicated antibodies of lysates derived from s.c. tumors from mice without dox in the drinking water (ΔEGFR-dependent) and escaper tumors that showed regrowth after a period of stasis after initial dox administration as in Fig. 3 (ΔEGFR-independent escapers). Analysis was performed at least three times for each antibody with consistent results.

Fig. 5.

Increased expression of SΔE-1/KLHDC8A in ΔEGFR-independent U373MG cells and GBMs and effect of knock-down on tumorigenicity. (A) Western blot analysis of lysates from ΔEGFR-dependent or -independent escaper tumors as in Fig. 4 with a polyclonal rabbit anti-SΔE-1 antibody. Cos7 cells transfected with SΔE-1 were used as a positive control for the antibody. (B Inset) Two tet-regulatable U373MG cell lines established from escaper tumors, Esc-0 and Esc-1, were infected with shRNA-expressing retroviruses targeting SΔE-1/KLHDC8A (No. 1 or No. 2), a nonspecific sequence (N.S.), or Luciferase (Luc), and stable cell lines were established. qPCR analysis of SΔE-1/KLHDC8A gene expression was performed in the cell lines expressing the indicated retroviral shRNA constructs. Each bar represents the mean ± SD of three replicates. y axis represents the relative gene expression level. These cells (3 × 10⁶) were injected s.c. into the flanks of nude mice fed with dox-containing drinking water. Tumor volumes were measured at the indicated times to generate the growth curve of tumors with knockdown of SΔE-1/KLHDC8A expression. n = 8 mice for all groups, mean ± SEM.