Patients with neurofibromatosis type 1 develop aggressive Schwann cell neoplasms known as malignant peripheral nerve sheath tumors (MPNSTs). Although tumor suppressor gene mutations play an important role in MPNST pathogenesis, it is likely that dysregulated signaling by as yet unidentified growth factors also contributes to the formation of these sarcomas. To test the hypothesis that neuregulin-1 (NRG-1) growth factors promote mitogenesis in MPNSTs, we examined the expression and action of NRG-1 in human MPNSTs and neurofibromas, the benign precursor lesions from which MPNSTs arise. Multiple a and b transmembrane precursors from the class II and III NRG-1 subfamilies are present in both tumor types. Neoplastic Schwann cells within these neoplasms variably express the erbB kinases mediating NRG-1 responses (erbB2, erbB3 and/or erbB4). Human MPNST cell lines (Mash-1, YST-1, NMS-2 and NMS-2PC cells) similarly coexpress multiple NRG-1 isoforms and erbB receptors. These MPNST lines are NRG-1 responsive and demonstrate constitutive erbB phosphorylation. Treatment with PD168393 and PD158780, two structurally and mechanistically distinct erbB inhibitors, abolishes erbB phosphorylation and reduces DNA synthesis in these lines. These findings suggest that autocrine and/or paracrine NRG-1/erbB signaling promotes neoplastic Schwann cell proliferation and may be an important therapeutic target in neurofibromas and MPNSTs.

Malignant peripheral nerve sheath tumors (MPNSTs) are the most common malignancy associated with neurofibromatosis type 1 (NF1). These Schwann cell lineage-derived sarcomas aggressively invade adjacent nerve and soft tissue, frequently precluding surgical resection. Little is known regarding the mechanisms underlying this invasive behavior. We have shown that MPNSTs express neuregulin-1 (NRG-1) β isoforms, which promote Schwann cell migration during development, and NRG-1α isoforms, whose effects on Schwann cells are poorly understood. Hypothesizing that NRG-1β and/or NRG-1α promote MPNST invasion, we found that NRG-1β promoted MPNST migration in a substrate-specific manner, markedly enhancing migration on laminin but not on collagen type I or fibronectin. The NRG-1 receptors erbB3 and erbB4 were present in MPNST invadopodia (processes mediating invasion), partially colocalized with focal adhesion kinase and the laminin receptor β1-integrin and coimmunoprecipitated with β1-integrin. NRG-1β stimulated human and murine MPNST cell migration and invasion in a concentration-dependent manner in three-dimensional migration assays, acting as a chemotactic factor. Both baseline and NRG-1β induced migration were erbB-dependent and required the action of MEK 1/2, SAPK/JNK, PI-3 kinase, Src family kinases and ROCK-I/II. In contrast, NRG-1α had no effect on the migration and invasion of some MPNST lines and inhibited the migration of others. While NRG-1β potently and persistently activated Erk 1/2, SAPK/JNK, Akt and Src family kinases, NRG-1α did not activate Akt and activated these other kinases with kinetics distinct from those evident in NRG-1β stimulated cells. These findings suggest that NRG-1β enhances MPNST migration and that NRG-1β and NRG-1α differentially modulate this process.

Sporadic and neurofibromatosis type 2-associated schwannomas contain a glial growth factor (GGF)-like activity that has been hypothesized to promote neoplastic Schwann cell mitogenesis. It is not known whether this GGF-like activity is neuregulin-1 (NRG-1), an epidermal growth factor (EGF)-related molecule that regulates the proliferation, survival, and differentiation of developing Schwann cells, the related factor NRG-2, or another NRG/EGF ligand. We report that neoplastic Schwann cells within schwannomas overexpress multiple alpha and beta transmembrane precursors from the class II and class III NRG-1 subfamilies. NRG-2 alpha and beta transcripts are similarly overexpressed in some tumors. Of the other 8 known NRG/EGF ligands, only heparin-binding EGF, epiregulin, and TGFalpha are detectable in schwannomas. Neoplastic Schwann cells almost uniformly express erbB2 and erbB3, 2 membrane receptor tyrosine kinases mediating NRG-1 and NRG-2 action. Expression of the NRG receptor erbB4 and EGF receptor is also evident in schwannomas, but is more limited, occurring in only a subset of these tumors. ErbB2, the preferred dimerization partner for all erbB kinases, is constitutively phosphorylated in schwannomas. These observations suggest that autocrine, paracrine, and/or juxtacrine NRG-1/NRG-2 signaling promotes schwannoma pathogenesis and that this signaling pathway may be an important therapeutic target in schwannomas.

Tamoxifen is widely used to treat estrogen receptor (ER)-positive breast cancer. Recent findings that tamoxifen and its derivative 4-dehydroxy-tamoxifen (OHT) can exert ER-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in ER-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNSTs) where estrogen is not involved. In this study, we gained insights into the ER-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated K-Ras degradation through autophagy induction, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by tamoxifen and OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation and survival, MPNST xenograft growth, and the mechanism by which tamoxifen impeded these processes. We found that 1-5 μM 4-hydroxy-tamoxifen induced MPNST cell death, whereas 0.01-0.1 μM 4-hydroxy-tamoxifen inhibited mitogenesis. Dermal and plexiform neurofibromas, MPNSTs, and MPNST cell lines expressed ERβ and G-protein-coupled ER-1 (GPER); MPNSTs also expressed estrogen biosynthetic enzymes. However, MPNST cells did not secrete 17β-estradiol, exogenous 17β-estradiol did not stimulate mitogenesis or rescue 4-hydroxy-tamoxifen effects on MPNST cells, and the steroidal antiestrogen ICI-182,780 did not mimic tamoxifen effects on MPNST cells. Further, ablation of ERβ and GPER had no effect on MPNST proliferation, survival, or tamoxifen sensitivity, indicating that tamoxifen acts via an ER-independent mechanism. Consistent with this hypothesis, inhibitors of calmodulin (trifluoperazine, W-7), another known tamoxifen target, recapitulated 4-hydroxy-tamoxifen effects on MPNST cells. Tamoxifen was also effective in vivo, demonstrating potent antitumor activity in mice orthotopically xenografted with human MPNST cells. We conclude that 4-hydroxy-tamoxifen inhibits MPNST cell proliferation and survival via an ER-independent mechanism. The in vivo effectiveness of tamoxifen provides a rationale for clinical trials in cases of MPNSTs.

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFβ3 mice) develop MPNSTs. To determine whether P(0)-GGFβ3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFβ3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFβ3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFβ3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFβ3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras and K-Ras are coexpressed with their activators, (guanine nucleotide exchange factors), in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades.

Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 (NF1) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J x SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P0-GGFβ3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1-regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P0-GGFβ3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to “unmask” tumorigenesis in these animals, we followed cohorts of inbred P0-GGFβ3;Nf1+/−, P0-GGFβ3; Trp53+/− and control (P0-GGFβ3, Nf1+/− andTrp53+/−) mice for 1 year. We found no reduction in survival or tumors in control and P0-GGFβ3;Nf1+/− mice. In contrast, P0-GGFβ3; Trp53+/− mice died on average at 226 days, with MPNSTs present in 95% of these mice. MPNSTs in inbred P0-GGFβ3; Trp53+/− mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II-III) than the major MPNSTs (WHO grade III-IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P0-GGFβ3; Trp53+/− mice represent a novel model of low to high grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.