ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma

Abstract

Vestibular schwannomas (VS) arising sporadically or in patients with neurofibromatosis type 2 (NF2) consistently lack expression of Merlin, a tumor suppressor. Conventional treatment options include surgery and radiotherapy but there is no validated medical option. Recent evidence suggests that Merlin deficiency may result in abnormal activation of receptor tyrosine kinases (RTKs) and downstream signaling, promoting tumor growth. Although small-molecule RTK inhibitors are widely available for clinical use, no such therapy has been validated in patients with VS. To screen for RTK activation, surgical VS specimens from patients with and without NF2 were analyzed by phospho-RTK profiling arrays. Downstream signaling pathway activation was analyzed by phospho-MAPK arrays. Activated RTKs and downstream kinases were validated immunohistochemically in corresponding formalin-fixed, paraffin-embedded tissues. Phospho-RTK arrays and immunohistochemistry showed consistent overexpression and activation of EGFR family receptors and evidence of ERK1/2 downstream signaling was observed in all samples analyzed (n = 11). Based on the findings, the small-molecule EGFR/ErbB2 kinase inhibitor lapatinib was selected for evaluation of target inhibition and treatment efficacy in our in vitro human schwannoma model. EGFR/ErbB2 targeted therapy with lapatinib inhibited ErbB2 phosphorylation and survivin upregulation, as well as downstream ERK1/2 and AKT activation, resulting in decreased proliferation. We conclude that EGFR family receptor activation is a consistent feature of both sporadic and NF2-related VS. Molecular targeted therapy with lapatinib downregulates survivin and has antiproliferative activity in a preclinical VS model. Based on these findings, a clinical trial with lapatinib for the treatment of VS is currently underway.

Fig. 1.

Phospho-RTK profiling arrays. Representative arrays from a VS sample (A) and a control sample (normal vestibular nerve, B), showing relative phosphorylation status of RTKs. Phosphorylation level of each RTK is represented by duplicate spots. Positive control spots are located in all 4 corners of the array. (1) EGFR, (2) ErbB2, (3) ErbB3, (4) AXL. Results from all samples are summarized in Table 1.

Fig. 2.

Phospho-MAPK profiling arrays. Representative arrays from a VS sample (A) and a control sample (normal vestibular nerve, B), showing relative phosphorylation status of MAP kinases and serine/threonine kinases. Phosphorylation level of each RTK is represented by duplicate spots. Positive control spots are located in 3 corners of the array. (1) ERK1, (2) ERK2. Results from all samples are summarized in Table 2.

Fig. 3.

Immunohistochemistry of VS specimens. Representative staining of VS for EGFR (A), ErbB2 (B), phospho-ERK1/2 (C), and survivin (D) is shown. Antigens are visualized by brown membranous and/or nuclear staining.

Fig. 4.

The effects of lapatinib on β-heregulin-mediated signaling and survivin expression in human primary schwannoma cells (NF2^−/−). (A) Lapatinib (10 µM) is a very potent inhibitor of ErbB2 receptor activity. (B, C, and D) Lapatinib (10 µM) completely inhibits β-heregulin (100 ng/ml) mediated ERK1/2, AKT, and S6 ribosomal protein phosphorylation/activation. The cells were starved for 24 hours before stimulation, pre-treated with lapatinib (10 µM) for 40 minutes, and stimulated with β-heregulin (100 ng/ml) for 5 minutes. The phosphorylated/activated levels of ErbB2 p-Tyr^1221/1222, ERK1/2 p-Thr¹⁸³/p-Tyr¹⁸⁵, AKT p-Ser⁴⁷³, and S6 ribosomal protein p-Ser^235/236 were detected by immunoblotting. (E) Lapatinib downregulates the expression of survivin in human primary schwannoma cells. The cells were serum starved for 24 hours and incubated with lapatinib for 72 hours. Day 1: the cells were pre-incubated for 40 minutes with 0.5 µM lapatinib, Day 2: 0.25 µM lapatinib was added, Day 3: 5 µM of lapatinib was added. The cells were lysed on day 4, and total levels of survivin were detected by immunoblotting using anti-survivin antibody. The data are corrected to a loading control RhoGDI and given as a % of basal (nonstimulated cells).

Fig. 5.

Lapatinib effects on the proliferation and viability of human primary schwannoma cells (NF2^−/−), as well as survivin expression. (A) Lapatinib (3 µM) completely inhibits β-heregulin (100 ng/ml) and forskolin (0.5 µM) mediated schwannoma proliferation. (B) Lapatinib at 3 µM has no significant effect on schwannoma cell viability. (C) Lapatinib at 5 and 10 µM significantly decreases schwannoma cell viability in a concentration-dependent manner after 24 hours treatment. In all experiments, the cells were cultured for 24 hours with or without β-heregulin (100 ng/ml) + forskolin (0.5 µM) and lapatinib. Total cell amount was estimated by nuclear staining with DAPI (1 µg/ml) and cell counting. The viability of the cells was estimated with PI (2.5 µg/ml) staining and cell counting. The data are corrected to a loading control RhoGDI and given as the % of basal (nonstimulated). In (A), the data are normalized to the basal levels (nonstimulated cells) and given as a % of basal (nonstimulated). In (B) and (C), the data are given as a % of total cell number.