This study demonstrates the dramatic effect of expressing EGFR in mouse Schwann cells. Peripheral and cranial nerves in CNP-hEGFR mice exhibit diffuse changes that parallel the hallmarks of human cutaneous and plexiform neurofibromas. These include increased endoneurial collagen matrix, dissociation of Schwann cells from axons, Schwann cell hypercellularity, and mast cell accumulation. Changes were within the perineurium, as is common in plexiform neurofibromas. While previous studies showed that EGFR is expressed in MPNST cells and some neurofibroma Schwann cells (DeClue et al., 2000
; Li et al., 2002
), EGFR expression might have correlated with tumor formation, rather than being a causative event. Strikingly, our data are consistent with a causative, and early, role for EGFR in progression to neurofibroma formation. EGFR overexpression is characteristic of, and contributes to, formation of many other human cancers. Therapeutic reagents that target EGFR are in clinical trials (Cunningham et al., 2004
; Noble et al., 2004
; Ranson et al., 2002
; Yarden, 2001
), and, based on our data, could be considered as candidate therapeutics for NF1.
EGFR is not normally expressed in peripheral nerve Schwann cells. We used the CNP promoter to drive hEGFR expression in Schwann cells and specifically localized hEGFR to glial cells in endoneurium. It was possible that hEGFR heterodimerization with Schwann cell-expressed ErbB2 might occur, but we failed to detect such heterodimers in adult nerves. We demonstrated EGFR ligand expression in peripheral nerve, and in wild-type Schwann cells. Based on these findings, it is likely that hEGFR expressed in Schwann cells homodimerize upon ligand stimulation, and become activated. Indeed, we detected phosphorylation of hEGFR in nerves.
This new model provided the opportunity to study early stages in neurofibroma formation, as pathology worsened with age and proximally to distally. Electron microscopy showed that abnormal axon-glial interactions were present by 2 months of age. Schwann cells wrapped progressively fewer axons, and ultimately dissociated from axons and wrapped collagen fibrils. Effects of hEGFR expression were significantly greater on nonmyelinating Schwann cells than on those associated with myelin, even though both Schwann cell types expressed hEGFR. This is consistent with enhanced sensitivity of the nonmyelinating Schwann cell to perturbed tyrosine kinase signaling downstream of hEGFR, driving abnormal neuron-glial interactions. Emphasizing a role for critical levels of tyrosine signaling in axon-glial interactions, Chen et al. (2003)
showed that expression of a dominant negative ErbB4 receptor signaling in adult nonmyelinating Schwann cells causes progressive sensory loss with disruption of nonmyelinating Schwann cell bundles. Loss of the adhesion molecule L1 might also be involved, as mice without L1 also show disrupted nonmyelinating axon-Schwann cell units (Haney et al., 1999
). Human neurofibromas preferentially arise from sensory nerves that contain mainly unmyelinated Schwann cells. Based on our results, many neurofibroma Schwann cells free of axons may have been associated at one time with small axons.
Mast cell accumulation and fibrosis are characteristic features of human neurofibromas and of peripheral nerves in hEGFR mice. Mast cells did not express hEGFR on Western blotting or immunostaining of tissue sections (data not shown). Perineurial cells, of mesodermal origin, were also negative for transgene expression. Nonglial cells accounted for few of the cells in the hEGFR nerves. It is reasonable to postulate that hEGFR signaling in Schwann cells stimulates production and secretion of mast cell chemoattractants, and here we have confirmed that mRNA encoding the mast cell chemoattractants BNDF, MCP-1, SCF, TGFβ1, and VEGF are each upregulated in mutant nerve; one or more may recruit mast cells to the nerve. Mast cells migrate to SCF secreted by Schwann cells (Yang et al., 2003
). Mast cell degranulation stimulates collagen deposition by fibroblasts. Fibrosis is commonly linked to mast cell degranulation (Krishnaswamy et al., 2001
). Thus, Schwann cell hEGFR expression could promote multicellular changes in peripheral nerve.
It remains possible that Schwann cell overexpression of tyrosine kinase receptors in addition to EGFR could drive the Schwann cell hyperplasia with fibrosis and mast cell accumulation documented. To our knowledge, to date, no other studies have examined transgenic overexpression of tyrosine kinase receptors in Schwann cells. However, the specificity of our result is suggested, in that Schwann cell transgenic overexpression of GGFβ3, a ligand for ErbB3, results in peripheral neuropathy, but not in the fibrosis and mast cell accumulation that we report (Huijbregts et al., 2003
). Prenatal treatment of mice with the chemical carcinogen ENU (N-nitrosoethylurea) causes activation of the ErbB2 tyrosine kinase in mouse Schwann cells (Buzard et al., 1999
). Such exposure induces invasive Schwann cell tumors with interdigitating Schwann cell processes, cyst formation, and parallel arrays of elongated neoplastic Schwann cells in about 4% of mice from sensitive strains (Wechsler et al., 1979
); C57Bl/6 mice, such as those in our study, are highly resistant. Carcinogen-induced mouse nerve tumors and growth factor overexpression result in nerve pathologies very different from those in the EGFR transgenic mice. Together with our study, these data strongly support a key role for tyrosine kinase pathways in regulation of non-myelin-forming Schwann cells in peripheral nerve.
To better understand the relevance of EGFR expression to malignant tumor formation, we mated Nf1+/–
mice to EGFRwa-2
/+ mice. In a mouse model of colon cancer, the wa-2 mouse decreased adenoma number and prolonged survival (Roberts et al., 2002
; Sibilia et al., 2000
). In this study, reduction in EGFR expression in Nf1+/–
/+ mice decreased tumor formation and mortality significantly. This data supports a crucial role for EGFR downstream of Nf
1 signaling. MPNST formation is rare in the CNPase-EGFR-expressing mice (shown here), as it is after chemical carcinogen exposure, affecting only 0.05% of sensitive mice (Wechsler et al., 1979
). It is likely that additional genetic events are necessary for frequent malignancy in the CNPase-hEGFR model. Other tyrosine kinases are expressed in human MPNST (Badache et al., 1998
) and could contribute to tumor growth. In human, EGFR is amplified in 26% of MPNST (Perry et al., 2002
), so this receptor is likely to be relevant to human MPNST tumor formation as it is in the Nf1
-driven tumor model studied here.
This study shows that EGFR expression in Schwann cells results in nerve hyperplasia with occasional neurofibroma formation. To develop neurofibromas at a higher rate, or malignancies, it may be necessary to cross these mice to strains with other mutations. Cre/lox mediated ablation of Nf1
in Schwann cells resulted in nerve pathology similar to that observed here, but only when the mice were also hemizygous for Nf1
mutation (Zhu et al., 2002
). Our model does not require mast cells and/ or fibroblasts to be Nf1+/–
, as crossing hEGFR to Nf1+/–
mice did not increase nerve size or Schwann cell number. The similar phenotype of hEGFR nerves to nerves in mice and humans, with loss of function mutation at Nf1
(Cichowski et al., 1999
; Vogel et al., 1999
; Zhu et al., 2002
), suggests that when loss of Nf1
predisposes Schwann cells, or their progenitors, to upregulate EGFR, neurofibroma formation ensues. This new model will allow testing of EGFR antagonists in vivo for their ability to prevent the formation of tumorgenic phenotypes. This study further validates EGFR as a potential target for therapeutic intervention in NF1 patients.