Currently, no medical therapies approved by the FDA are available for sporadic and NF2-associated VS. Although observation with serial imaging, microsurgical resection, and stereotactic radiotherapy provide reasonable management options, potent, effective, and non-toxic medications that inhibit VS progression would greatly benefit VS patients. Further characterization of key signaling pathway and preclinical drug testing are vital in exploring chemotherapeutic options for VS. The current study examines the expression of total and phosphorylated ErbB receptors and their in vitro response to inhibitors. We demonstrated consistently higher levels of total and phosphorylated ErbB3 in VS tumor tissues relative to paired vestibular nerves, while both phospho-EGFR and phospho-ErbB3 were elevated in cultured VS cells. Furthermore, VS cell proliferation was inhibited by ErbB receptor inhibitors, and Erlotinib exhibited higher potency of growth inhibition than Lapatinib.
The role and mechanism of ErbB-family receptors in VS development and progression has not been fully elucidated; however activation or overexpression ErbB receptors has been linked to increased Schwann cell proliferation and VS tumor formation (28
). Many other human cancers overexpress ErbB receptors (31
). EGFR and ErbB4 are fully functional RTKs while ErbB2 does not bind any known ligand. ErbB3 lacks independent kinase activity; however, on heterodimerization with other ErbB members, the cytoplasmic domain of phosphorylated and activated ErbB3 potently recruits PI3K to six distinct phosphotyrosine residues on ErbB3. Phospho-ErbB3 efficiently evades ligand-induced degradation (36
) while strongly activating the pro-growth AKT/PI3K pathway, especially when bound to ErbB2 (38
). ErbB2 heterodimers are characterized by high affinity, broad specificity, and potent mitogenic signaling potential due to frequent recycling back to the plasma membrane after internalization.
To better understand merlin’s relationship with RTKs in Schwann cells, Lallemand et al. (15
) cultured primary murine Schwann cells derived from Nf2flox2/flox2
) and utilized adenovirus-mediated Cre expression to generate two distinct populations of Schwann cells, namely Nf2+/+
. While no differences were observed in growth rates between Nf2+/+
Schwann cells in sub-confluent cultures, the Nf2−/−
Schwann cells were not able to sense contact inhibition and kept growing despite confluence. Nf2+/+
Schwann cells became senescent after five to seven passages in culture while Nf2−/−
Schwann cells did not undergo replicative-senescence. The loss of merlin increased the abundance of ErbB2, ErbB3, insulin-like growth factor 1 receptor (IGF1R), and PDGFR-β at the plasma membrane in confluent but not sub-confluent Schwann cells. Reintroduction of merlin into Nf2−/−
SCs reduced recycling of internalized growth factor receptors back to the plasma membrane in confluent cells. Varying the concentration of insulin, an IGF1R ligand, had no effect on the Nf2−/−
Schwann cell phenotype, but lowering the levels of heregulin-β1, an ErbB receptor ligand, restored contact inhibition and replicative-senescence, suggesting that ErbB receptor signaling contributed directly to the deregulated growth observed in Nf2
-deficient cells. As VS are merlin-deficient, they often display aberrant ErbB receptor signaling. Consistent with this notion, we observed elevated ErbB receptor expression, particularly ErbB3, in both VS tumor and cultured cells. However, cultured VS cells also showed high levels of phospho-EGFR expression, suggesting that culture conditions selectively enhance EGFR activation and signaling.
Studies using human tissues have found that EGFR and ErbB2 are upregulated in VS and may be targets for therapeutic intervention. Doherty and colleagues (23
) demonstrated that VS upregulated EGFR in 68% and ErbB2 in 84% of specimens. EGF was upregulated in all NF2-related VS, but none of the sporadic VS, and heregulin, an ErbB ligand, was upregulated in 86% of sporadic VS but only 19% of NF2-related VS. Using cultured VS cells, Brown and Hansen (22
) found that phosphorylated ErbB2 localized to lipid rafts, micro-domains in the plasma membrane that regulate receptor signaling. Our results on phosphorylated ErbB receptor expression are consistent with a recent report by Ammoun et al. who showed elevated expression of multiple phosphorylated ErbB-family receptors in VS tumors (41
As reported previously, we demonstrated activation of multiple RTKs in VS compared to paired vestibular nerves. Although the number of tumor/nerve pairs used in this study is limited, our data represents a unique within-patient comparison that has not been described previously. Based on the current evidence, a larger study to compare paired samples is warranted. Interestingly, although all three sporadic VS tumors exhibited some variability of phosphor-ErbB receptor expression, they consistently expressed total and phospho-ErbB3. In addition, we observed that one NF2 tumor (VS1 from ) had expression of all four ErbB members with prominent ErbB3 staining, consistent with the results from phospho-RTK arrays and Western blot analysis.
In addition to phospho-ErbB receptors, we identified elevated expression of phosphorylated FGFR-2α, insulin receptor, macrophage-stimulating protein receptor (MSPR), PDGFR-β, C-RET, and EphA4 in VS. Activation of FGFR and PDGFR has been linked to VS growth and progression (43
). While the remaining phospho-RTKs have been linked to human cancers, their roles in VS tumorigenesis are presently unknown.
Most of the currently-available drugs that inhibit the ErbB family of receptors target EGFR and ErbB2. Inhibition of EGFR with Gefitinib has been shown to induce a cytostatic effect in merlin-deficient cells (14
). Trastuzumab, a monoclonal antibody to ErbB2, has been shown to inhibit growth of VS cells (45
). Clinical use of Erlotinib was reported in one patient with VS, and a reduction of tumor volume was observed in this short-term study (46
). However, in a subsequent study with 11 NF2 patients, only a small subset of Erlotinib-treated patients had prolonged stable disease (47
). A clinical trial for Lapatinib is ongoing but the results have not been reported. Our data showed that Lapatinib was less potent than Erlotinib in suppressing schwannoma cell proliferation. While the reason for this finding is presently not understood, it is plausible that inhibition of ErbB2 by Lapatinib may result in up-regulation of ErbB3 as reported by Garrett et al. (42
). Inhibition of both ErbB2 and ErbB3 is likely to be a more effective therapeutic strategy in VS. We are presently investigating this possibility.
It should be mentioned that the response of cells to ErbB inhibitors may be cell type and context-dependent. Frolov et al. (48
) found that ErbB3:EGFR heterodimerization induced activation of the PI3K/AKT pathway, and ErbB3 expression increased susceptibility of pancreatic cancer cells to Erlotinib treatment. Liles et al. (49
) found that combined treatment with MM-121, an ErbB3 monoclonal antibody, and Erlotinib gave rise to a greater degree of tumorigenesis inhibition in pancreatic ductal adenocarcinoma cells that depend on ErbB3-mediated signaling. In breast cancer cells that are driven by ErbB2 activation, the presence of ErbB3 has been shown to promote growth and may overcome RTK inhibition by EGFR and ErbB2 inhibitors (50
). Furthermore, ErbB inhibitor efficacy appears to be closely linked to ErbB3 transphosphorylation. We (51
) previously reported activation of the PI3K/AKT pathway in VS cells. It is tempting to speculate that the interaction with ErbB3 may be significant in this process. Although Erlotinib is thought to primarily target EGFR, our data showed that Erlotinib could decrease phosphorylation of multiple ErbB receptors in schwannoma cells. It will be interesting to examine whether this decrease is due to the transphosphorylation activity of EGFR.
This study supports a need for further development of a more potent ErbB receptor inhibitor and a combined treatment strategy for VS. ErbB receptor inhibitors have the advantage of a more favorable clinical side effect profile than other chemotherapeutics in long-term dosing (52
), which would be required in patients with VS. A safe and effective medical treatment, which preserves neurologic function while inhibiting VS tumor growth, would be most welcomed by the patients, their loved ones and their treating physicians alike.