Actin-rich protrusions and nonlocalized GTPase activation in Merlin-deficient schwannomas.

Schwannomas lack both alleles for the tumor suppressor Merlin, a cytoskeleton-membrane linker. Previous results showed increased cell spreading of schwannoma cells, but little is known about the underlying mechanisms. Electron microscopy reveals that schwannoma cells not only show more lamellipodia/ruffles but also multiple filopodia. We show that Cdc42, important in filopodia formation, is activated. Both Rac1 and Cdc42 are found all around the cell periphery and in colocalization with their effector phospho-p21 activated kinase in human schwannoma cells. We therefore claim that Rac1 and Cdc42 are activated in a nonlocalized manner, which explains the disperse distribution of lamellipodia/ruffles and filopodia. Using live cell imaging, we further demonstrate continuous remodeling of the many actin-rich protrusions in schwannoma cells. The underlying cytoskeleton of these structures is thin and extensively branched. The actin-related protein 2/3 complex, a major regulator of actin branching, is enriched in the many lamellipodia and ruffles of human primary schwannoma cells. We suggest that the Merlin deficiency in human primary schwannoma cells leads to a random, nonlocalized activation of Rac1 and Cdc42, inducing many actin-rich protrusion zones, not only at the leading edge but also all around the cell periphery. Their nondirectional occurrence together with the continuous and highly dynamic actin remodeling results in the dedifferentiation of these tumor cells.

Altered adhesive structures and their relation to RhoGTPase activation in merlin-deficient Schwannoma.

Schwannomas are Schwann cell tumors of the nervous system that occur spontaneously and in patients with neurofibromatosis 2 (NF2) and lack the tumor suppressor merlin. Merlin is known to bind paxillin, beta1 integrin and focal adhesion kinase, members of focal contacts, multi-protein complexes that mediate cell adhesion to the extracellular matrix. Moreover, merlin-deficient Schwannomas show pathological adhesion to the extracellular matrix making the characterization of focal contacts indispensable. Using our Schwannoma in vitro model of human primary Schwann and Schwannoma cells, we here show that Schwannoma cells display an increased number of mature and stable focal contacts. In addition to an involvement of RhoA signaling via the Rho kinase ROCK, Rac1 plays a significant role in the pathological adhesion of Schwannoma cells. The Rac1 guanine exchange factor- beta-Pix, localizes to focal contacts in human primary Schwannoma cells, and we show that part of the Rac1 activation, an effect of merlin-deficiency, occurs at the level of focal contacts in human primary Schwannoma cells. Our results help explaining the pathological adhesion of Schwannoma cells, further strengthen the importance of RhoGTPase signaling in Schwannoma development, and suggest that merlin's role in tumor suppression is linked to focal contacts.

PAK kinase regulates Rac GTPase and is a potential target in human schwannomas.

Merlin loss causes benign tumours of the nervous system, mainly schwannomas and meningiomas. Schwannomas show enhanced Rac1 and Cdc42 activity, the p21-activated kinase 2 (PAK2) activation and increased ruffling and cell adhesion. PAK regulates activation of merlin. PAK has been proposed as a potential therapeutic target in schwannomas. However where PAK stands in the Rac pathway is insufficiently characterised. We used a novel small-molecule PAK inhibitor, IPA-3, to investigate the role of PAK activation on Rac1/Cdc42 activity, cell spreading and adhesion in human primary schwannoma and Schwann cells. We show that IPA-3 blocks activation of PAK2 at Ser192/197 that antagonises PAK's interaction with Pix. Accordingly, Pix-mediated Rac1 activation is decreased in IPA-3 treated schwannoma cells, indicating that PAK acts upstream of Rac. We show that this Rac activation at the level of focal adhesions in schwannoma cells is essential for cell spreading and adhesion in Schwann and schwannoma cells.

Dissecting and targeting the growth factor-dependent and growth factor-independent extracellular signal-regulated kinase pathway in human schwannoma.

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 beta (PDGFRbeta), 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 PDGFRbeta-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 PDGFRbeta-mediated 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.