Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Abstract

Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor B (PDGFRB), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRB-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRBmediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials. [Cancer Res 2008;68(13):5236-45]

Insulin-like growth factor-binding protein-1 (IGFBP-1) regulates human schwannoma proliferation, adhesion and survival

“…Complete culture medium (GFM) (upper panel) or serum-free medium (DMEM, middle pane) collected from schwannoma cells and cell lysates were treated with lambda phosphatase and levels of phosphorylated IGFBP-1 in medium and phosphorylated ERK1/2 in cell lysates (positive control for phosphatase activity) were monitored by western blotting (bottom panel). Short-term stimulation with recombinant IGFBP-1 upregulates phosphorylation of FAK but not ERK, AKT and JNK in b1 integrin-mediated manner Next, we investigated the effects of IGFBP-1 on FAK, ERK, AKT and c-Jun N-terminal kinase (JNK) phosphorylation previously shown to be strongly activated in basal conditions in schwannoma cells Ammoun et al, 2008). In order to have well-controlled system, we starved cells for 24 h, then medium was changed to fresh DMEM for 30 min to minimize autocrine-mediated signalling and cells were stimulated with recombinant IGFBP-1.…”

“…As shown in other cancer models IGFBP-1, through its COOHterminal RGD motif (Jones et al, 1993b;Yee, 2002), can act via b1-integrin toward FAK (Zumkeller and Westphal, 2001;Zhang and Yee, 2006;Perks et al, 2007) both strongly overexpressed and basally activated in schwannoma Ammoun et al, 2008) and may potentiate cell proliferation, migration and survival (Giancotti and Ruoslahti, 1999). Therefore, we wondered what role IGFBP-1 has in schwannoma development.…”

“…To study schwannoma pathobiology and define therapeutic targets, we used our human in vitro model for schwannoma. We showed that plateletderived growth factor receptor b (PDGFRb) and ErbB2/3 are overexpressed/activated in schwannoma in vitro and in vivo leading to strong/long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT (Fraenzer et al, 2003;Ammoun et al, 2008Ammoun et al, , 2010aHilton et al, 2009). ERK1/2 activation increases schwannoma proliferation (Ammoun et al, 2008(Ammoun et al, , 2010b.…”

“…We showed that plateletderived growth factor receptor b (PDGFRb) and ErbB2/3 are overexpressed/activated in schwannoma in vitro and in vivo leading to strong/long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT (Fraenzer et al, 2003;Ammoun et al, 2008Ammoun et al, , 2010aHilton et al, 2009). ERK1/2 activation increases schwannoma proliferation (Ammoun et al, 2008(Ammoun et al, , 2010b. Survival is also augmented in schwannoma (Utermark et al, 2005) because of the activity of ErbB2/3 and survivin (Ammoun et al, 2010a).…”

“…Pathologically increased cell-matrix adhesion in schwannoma is dependent on overexpressed/activated integrins but is completely independent of PDGFRb, suggesting that other factors are involved Ammoun et al, 2008). Furthermore, schwannoma cells display elevated basal (non-stimulated) activity of ERK1/2, AKT and focal-adhesionkinase (FAK) (Ammoun et al, 2008) and increased basal proliferation, which is only partly dependent on PDGFRb (Ammoun et al, 2008) and is independent of ERK1/2 and ErbB2/3 (Ammoun et al, 2008(Ammoun et al, , 2010a suggesting the involvement of other important factors. Our aim was to identify targets relevant in basal proliferation and cell-matrix adhesion of schwannomas.…”