Schwannoma Cells Research Articles

Targeting ERK1/2 activation and proliferation in human primary schwannoma cells with MEK1/2 inhibitor AZD6244

Deficiency of the tumor suppressor merlin leads to the development of multiple tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas. Due to the benign character of these tumors, classical chemotherapy is ineffective. Current therapies, surgery, and radiosurgery are local and quite invasive, thus new systemic treatments are required. We have previously described the Raf/mitogen-activated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation and its role in schwannoma growth. Here, we targeted MEK1/2 known as a convergence point for multiple cascades towards ERK1/2 activation and cell proliferation, using MEK1/2 inhibitor AZD6244 (ARRY-142886; Astra Zeneca).We show that AZD6244 at low concentration completely abolished platelet-derived growth factor-DD-mediated ERK1/2 activation and cell proliferation in human primary schwannoma cells. Moreover, this drug was not toxic for either schwannoma or Schwann cells and has been reported to be safe with tolerable side effects. Thus, AZD6244 can be considered as a drug candidate for schwannoma treatment.

Human Schwannomas Express Activated Platelet-Derived Growth Factor Receptors and c-kit and Are Growth Inhibited by Gleevec (Imatinib Mesylate)

Schwannomas, although benign, can be fatal or give rise to significant morbidity due to an unpredictable growth rate. They can reoccur after surgery or radiation, current treatments each with significant inherent risks. These risks are further amplified in neurofibromatosis type 2 (NF2), a germ line predisposition syndrome characterized by multiple schwannomas, underlying the need for biological targeted therapies. Gleevec (STI571, imatinib mesylate), in addition to the bcr-abl oncogene in chronic myelogenous leukemia, inhibits c-kit and platelet-derived growth factor receptor (PDGFR) signaling, thereby expanding its use to several malignant and benign human diseases. In the present study, we show that human sporadic and NF2-associated schwannomas have increased expression along with activation of PDGFR-alpha, PDGFR-beta, and c-kit receptors, compared with normal or traumatic nerve. Using the human NF2-null HEI-193 schwannoma cell line, Gleevec inhibited schwannoma viability, proliferation, and anchorage-independent growth, as well as induced apoptosis in a dose-dependent manner (IC(50) 5-10 micromol/L). These antitumorigenic effects were correlated to inhibition of PDGFR-alpha, PDGFR-beta, and c-kit activation/phosphorylation and major downstream signaling pathways. Lack of robust xenograft or transgenic models of schwannomas prevents extension of these studies in vivo. However, the established long track record and tolerable toxicity of Gleevec already in clinical use and our preclinical data lead us to propose that Gleevec should be evaluated in human schwannomas with shown progressive growth.

Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T Schwannoma cells

Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. In many cell types, a fraction of sphingolipids contain 2-hydroxy fatty acids, produced by fatty acid 2-hydroxylase (FA2H), as the N-acyl chain of ceramide [hydroxyl fatty acid (hFA)-sphingolipids]. FA2H is highly expressed in myelin-forming cells of the nervous system and in epidermal keratinocytes. While hFA-sphingolipids are thought to enhance the physical stability of specialized membranes produced by these cells, physiological significance of hFA-sphingolipids in many other cell types is unknown. In this study, we report novel roles for FA2H and hFA-sphingolipids in the regulation of the cell cycle. Treatment of D6P2T Schwannoma cells with dibutyryl-cAMP (db-cAMP) induced exit from the cell cycle with concomitant upregulation of FA2H. Partial silencing of FA2H in D6P2T cells resulted in 60-70% reduction of hFA-dihydroceramide and hFA-ceramide, with no effect on nonhydroxy dihydroceramide and ceramide. Under these conditions, db-cAMP no longer induced cell cycle exit, and cells continued to grow and divide. Immunoblot analyses revealed that FA2H silencing prevented db-cAMP-induced upregulation of cyclin-dependent kinase inhibitors p21 and p27. These results provide evidence that FA2H is a negative regulator of the cell cycle and facilitates db-cAMP-induced cell cycle exit in D6P2T cells.

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.

Growth inhibitory and anti-tumour activities of OSU-03012, a novel PDK-1 inhibitor, on vestibular schwannoma and malignant schwannoma cells

Background Vestibular schwannomas (VS) frequently express high levels of activated AKT. Small-molecule inhibitors of AKT signalling may have therapeutic potential in suppressing the growth of benign VS and malignant schwannomas. Method Primary VS and Schwann cells, human malignant schwannoma HMS-97 cells and mouse Nf2 –/– Schwann cells and schwannoma cells were prepared to investigate the growth inhibitory and anti-tumour activities of OSU-03012, a celecoxib-derived small-molecule inhibitor of phosphoinositide-dependent kinase-1. Cell proliferation assays, apoptosis, Western blot, in vivo xenograft analysis using SCID mice and immunohistochemistry were performed. Results OSU-03012 inhibited cell proliferation more effectively in both VS and HMS-97 cells than in normal human Schwann cells. The IC 50 of OSU-03012 at 48 h was approximately 3.1 μM for VS cells and 2.6 μM for HMS-97 cells, compared with the IC 50 of greater than 12 μM for human Schwann cells. Similarly, mouse Nf2 –/– schwannoma and Nf2 –/– Schwann cells were more sensitive to growth inhibition by OSU-03012 than wild-type mouse Schwann cells and mouse schwannoma cells established from transgenic mice carrying the NF2 promoter-driven SV40 T-antigen gene. Like VS cells, malignant schwannoma HMS-97 cells expressed high levels of activated AKT. OSU-03012 induced apoptosis in both VS and HMS-97 cells and caused a marked reduction of AKT phosphorylation at both the Ser-308 and Thr-473 sites in a dose-dependent manner. In vivo xenograft analysis showed that OSU-03012 was well tolerated and inhibited the growth of HMS-97 schwannoma xenografts by 55% after 9 weeks of oral treatment. The anti-tumour activity correlated with reduced AKT phosphorylation. Conclusion OSU-03012 is a potential chemotherapeutic agent for VS and malignant schwannomas.

Measurement Of Hydrogen Ion Activity In The Intercellular Space Of Schwannoma Tumors

We are exploring the use extracellular fluorescent indicators to measure analytes in the microdomains between adjacent cells in tissues. Our primary initial goal is to measure changes in H+ activity in the intercellular space of xenograft tumor models before and after drug treatment. It is thought that acidification in the core of tumors protects these cancers from the primarily, weak base, chemotherapeutic drugs. We are comparing the use of functionalized nanoparticles and expressible indicators as putative extracellular sensors. Functionalized, fluorescein-labeled nanoparticles were constructed that enable the particles to adhere to the surface of schwannoma cells. However, preliminary work with the nanoparticles showed toxic side effects, at least over a period of days. We have also been exploring the use of expressible membrane bound H+ indicators, glycosylphosphatidylinositol (GPI)-anchored GFP mutants. A problem with these indicators is that they localize to the plasma membrane and also the entire exocytotic pathway. In vitro testing of the indicators was accomplished by aggregating single cells into clusters (artificial tumors) using dielectrophoresis (DEP) and agarose molds. Preliminary results indicated that nanoparticles could be trapped within artificial tumors using DEP as long as electric fields are applied. Long term (7 days) intercellular pH sensing was achieved in vitro using GPI-GFP expressing schwannoma cells in an artificial tumor crafted with an agarose mold. While we are currently exploring the use of sensors to measure changes in pH in the intercellular space, we are also investigating the development and testing of other sensors to detect analytes such as ATP, Cl-, Na+, and K+. This will provide valuable information regarding cellular defenses against chemotherapeutic drugs and mechanisms of therapeutic drug action.This project was funded by the NIH:NCRR as a national research resource [P41 RR001395]

Interleukin‐6 is required for the early induction of glial fibrillary acidic protein in Schwann cells during Wallerian degeneration

Signal transducer and activator of transcription 3 (STAT3) regulates gene transcription in response to cytokines and growth factors. In the central nervous system, STAT3 plays a role in neuroprotection and reactive gliosis after lesions. During peripheral nerve regeneration, a nerve injury-induced up-regulation of cytokines and growth factors accompanies STAT3 activation in sensory neurons and Schwann cells (SCs) even though its molecular details and functions are unknown. We then analyzed the ligands and functions of STAT3 activation in RT4 schwannoma cells and adult SCs in vitro and in vivo. We have identified that interleukin-6 (IL-6), but not ciliary neurotrophic factor, leukemia inhibitory factor, or ligands for receptor tyrosine kinases, activates STAT3 in SCs. The IL-6/STAT3 signaling in primary SCs and RT4 cells induced the gene expression of glial fibrillary acidic protein (GFAP), which is known to be required for the proper regeneration of the injured nerves. Finally, the GFAP induction in the sciatic nerves after injury was significantly delayed in IL-6-deficient mice. These findings indicate that IL-6 plays an important role in STAT3-dependent GFAP induction in SCs during peripheral nerve regeneration.

Bioluminescent Imaging of Intracranial Vestibular Schwannoma Xenografts in NOD/SCID Mice

Intracranial vestibular schwannoma xenografts can be successfully established and followed with bioluminescent imaging (BLI). Transgenic and xenograft mouse models of vestibular schwannomas have been previously reported in the literature. However, none of these models replicate the intracranial location of these tumors to reflect the human disease. Additionally, traditional imaging methods (magnetic resonance imaging, computed tomography) for following tumor engraftment and growth are expensive and time consuming. BLI has been successfully used to longitudinally follow tumor treatment responses in a noninvasive manner. BLI's lower cost and labor demands make this a more feasible approach for tumor monitoring in studies involving large numbers of mice. Patient excised vestibular schwannomas were cultured and transduced with firefly luciferase expressing lentivirus. One million cells were stereotactically injected into the right caudate nucleus of 21 nonobese diabetic/severe combined immunodeficient mice. Schwannoma engraftment and growth was prospectively followed for 30 weeks after injection with BLI. After animal sacrifice, the presence of human tumor cells was confirmed with fluorescent in situ hybridization. Eight (38%) of 21 mice successfully engrafted the schwannoma cells. All of these mice were generated from 4 (67%) of the 6 patient excised tumors. These 8 mice could be differentiated from the nonengrafted mice at 21 weeks. The engrafted group emitted BLI of greater than 100,000 photons/s (range, 142,478-3,106,300 photons/s; average, 618,740 photons/s), whereas the nonengrafted group were all under 100,000 photons/s (range, 0-76,010 photons/s; average, 10,737 photons/s) (p < 0.001). Fluorescent in situ hybridization analysis confirmed the presence of viable human schwannoma cells in much greater numbers in those mice with stable or growing tumors compared with those whose tumors regressed. We have successfully established an intracranial schwannoma xenograft model that can be followed with noninvasive BLI. We hope to use this model for in vivo testing of schwannoma tumor therapies.

A novel mechanism of methylglyoxal cytotoxicity in neuroglial cells

Methylglyoxal (MGO) is an endogenous dicarbonyl compound that is highly produced in hyperglycemic conditions. It forms advanced glycation endproducts that are believed to contribute, as etiological factors, to the pathophysiology of diabetic complications. In addition, MGO suppresses cell viability through the induction of apoptosis in vitro. In this study, we have, for the first time, demonstrated the effect of MGO on the gp130 cytokine-induced signal transducer and activator of transcription 3 (STAT3) responses in RT4 schwannoma, PC12 pheochromocytoma and U87MG glioma cells. At dose that very mildly affects cell viability, MGO rapidly induces endocytotic degradation of gp130, which involves the di-leucine internalization motif in the cytoplasmic domain of gp130, without affecting other growth factor receptors. Concomitant inhibition of basal and interleukin-6-induced STAT3 activation was observed following pre-treatment with MGO. The inhibitory effect of MGO on the gp130/STAT3 signaling was prevented by the pre-treatment with an advanced glycation endproduct scavenger aminoguanidine. Finally, these deleterious effects of MGO on STAT3 signaling led to down-regulation of a STAT3 target gene, Bcl-2, and sensitized cellular toxicity induced by H(2)O(2) and etoposide. Our data indicate that MGO affects cell viability via desensitization of gp130/STAT3 signaling, which is the key signaling pathway for cell survival, and thereby promotes cytotoxicity.

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.

272 POSTER Activity of the anti-cancer aptamer AS1411 includes regulation of Bcl-2 family members

Vestibular schwannomas (VS) frequently express high levels of activated AKT. Small-molecule inhibitors of AKT signalling may have therapeutic potential in suppressing the growth of benign VS and malignant schwannomas.Primary VS and Schwann cells, human malignant schwannoma HMS-97 cells and mouse Nf2–/– Schwann cells and schwannoma cells were prepared to investigate the growth inhibitory and anti-tumour activities of OSU-03012, a celecoxib-derived small-molecule inhibitor of phosphoinositide-dependent kinase-1. Cell proliferation assays, apoptosis, Western blot, in vivo xenograft analysis using SCID mice and immunohistochemistry were performed.OSU-03012 inhibited cell proliferation more effectively in both VS and HMS-97 cells than in normal human Schwann cells. The IC50 of OSU-03012 at 48 h was approximately 3.1 μM for VS cells and 2.6 μM for HMS-97 cells, compared with the IC50 of greater than 12 μM for human Schwann cells. Similarly, mouse Nf2–/– schwannoma and Nf2–/– Schwann cells were more sensitive to growth inhibition by OSU-03012 than wild-type mouse Schwann cells and mouse schwannoma cells established from transgenic mice carrying the NF2 promoter-driven SV40 T-antigen gene. Like VS cells, malignant schwannoma HMS-97 cells expressed high levels of activated AKT. OSU-03012 induced apoptosis in both VS and HMS-97 cells and caused a marked reduction of AKT phosphorylation at both the Ser-308 and Thr-473 sites in a dose-dependent manner. In vivo xenograft analysis showed that OSU-03012 was well tolerated and inhibited the growth of HMS-97 schwannoma xenografts by 55% after 9 weeks of oral treatment. The anti-tumour activity correlated with reduced AKT phosphorylation.OSU-03012 is a potential chemotherapeutic agent for VS and malignant schwannomas.

Validation of the p21-Activated Kinases as Targets for Inhibition in Neurofibromatosis Type 2

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlin's tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2(BBA) (NIH3T3/NF2(BBA)), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer.

Construction of eukaryotic expression vectors of two NF2 mutants and their expression in rat schwannoma cell line

Objective:To construct the eukaryotic expression vectors of two NF2 mutants(neurofibromatosisⅡ)and to study their expression in rat schwannoma cell line(RT4).Methods:Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Lys42 in NF2 into Pro in pcDNA3.1-NF2~(△Lys42Pro),and convert the codons for Phe 47 into that of Leu.RT4 cells were transiently transfected with the 3 kinds of plasmids containing the mutations and wide-type NF2 via lipofectin separately,then the expression levels of NF2 mRNA and protein were determined by RT-PCR and Western blotting in 3 groups.The cell proliferation was determined by the MTT after transfection.Results:DNA sequence analysis demonstrated the two-step mutagenesis was successful and the two plasmids of pcDNA3.1-NF2~(△ys42Pro)and pcDNA3.1-NF2~(△Phe47Leu)were obtained, both of the transfectants could produce merlin protein and mRNA.The two mutants had a significantly lower inhibitory rate for RT4 cells compared with wide-type NF2(P0.05).Conclusion:The recombinant plasmids pcDNA3.1-NF2~(△Lys42Pro)and pcDNA3.1-NF2~(△Phe47Leu)have been successfully constructed and they can be efficiently expressed in RT4 cells.

PACAP and VIP prevent apoptosis in schwannoma cells

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann 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.

Fowler Award Presentation: Effects of ErbB2 Signaling on the Response of Vestibular Schwannoma Cells to γ‐Irradiation

AbstractObjective: For vestibular schwannomas (VSs) that require treatment, options are limited to microsurgery or irradiation (IR). Development of alternative therapies that augment or replace microsurgery or IR would benefit patients not suitable for current therapies. This study explored the ability of ErbB2 inhibitors to modulate the effects of IR on VS cells.Study Design: Prospective study using primary cultures derived from human VSs.Methods: Primary cultures of VS cells were derived from acutely resected tumors. Cultures received single escalating doses (15–40 Gy) of γ‐irradiation from a 137Cs γ‐irradiation source. Cell proliferation was determined by BrdU uptake and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Trastuzumab (Herceptin) and PD158780 were independently used to inhibit ErbB2 signaling while neuregulin‐1β (NRG‐1) was used to activate ErbB2.Results: IR induces VS cell cycle arrest and apoptosis in doses greater than 20 Gy, demonstrating that VS cells are relatively radioresistant. This radioresistance likely arises from their low proliferative capacity as a sublethal dose of IR (10 Gy) strongly induces deoxyribonucleic acid (DNA) damage evidenced by histone H2AX phosphorylation. Inhibition of ErbB2, which decreases VS cell proliferation, protects VS cells from radiation‐induced apoptosis, while NRG‐1, an ErbB2 ligand and VS cell mitogen, increases radiation‐induced VS cell apoptosis.Conclusions: Compared with many neoplastic conditions, VS cells are relatively radioresistant. The radio‐protective effect of ErbB2 inhibitors implies that the sensitivity of VS cells to IR depends on their proliferative capacity. These results hold important implications for current and future treatment strategies.

Overexpression of CXCL16 and its receptor CXCR6/Bonzo promotes growth of human schwannomas

Chemokines and their receptors play a decisive role in tumor progression and metastasis. Here, we describe the expression of the CXCL16-CXCR6-system in human schwannomas of different localization and in malignant peripheral nerve sheath tumors. The transmembrane chemokine CXCL16 and its receptor CXCR6/Bonzo were overexpressed on the mRNA and protein levels in all tumor samples investigated as compared with normal peripheral or 8th cranial nerve tissues. Chromogenic immunostaining and confocal laser microscopy revealed that CXCL16 and CXCR6 were localized mainly on S-100 positive schwannoma cells. Cultured schwannoma cells responded to CXCL16-stimulation by phosphorylation of kinases p42/44 (Erk 2/1) that could be inhibited by the MEK1/2-inhibitor U0126 indicating an involvement of the mitogen-activated protein kinase signal transduction pathway. As a biological response, CXCL16 increased proliferation and induced migration of schwannomas. Hence, CXCL16 appears to be a novel growth factor for schwannomas of different localization.

Impaired intercellular adhesion and immature adherens junctions in merlin‐deficient human primary schwannoma cells

Schwannomas that occur spontaneously or in patients with neurofibromatosis Type 2, lack both alleles for the tumor suppressor and plasma membrane-cytoskeleton linker merlin. We have shown that human primary schwannoma cells display activation of the RhoGTPases Rac1 and Cdc42 which results in highly dynamic and ongoing protrusive activity like ruffling. Ruffling is an initial and temporally limited step in the formation of intercellular contacts like adherens junctions that are based on the cadherin-catenin system. We tested if there is a connection between Rac1-induced ongoing ruffling and the maintenance, stabilization and functionality of adherens junctions and if this is of relevance in human, merlin-deficient schwannoma cells. We show intense ongoing ruffling is not limited to membranes of single human primary schwannoma cells, but occurs also in membranes of contacting cells, even when confluent. Live cell imaging shows that newly formed contacts are released after a short time, suggesting disturbed formation or stabilization of adherens junctions. Morphology, high phospho-tyrosine levels and cortactin staining indicate that adherens junctions are immature in human primary schwannoma cells, whereas they display characteristics of mature adherens junctions in human primary Schwann cells. When merlin is reintroduced, human primary schwannoma cells show only initial ruffling in contacting cells and adherens junctions appear more mature. We therefore propose that ongoing Rac-induced ruffling causes immature adherens junctions and leads to impaired, nonfunctional intercellular adhesion in aggregation assays in merlin-deficient schwannoma cells that could be an explanation for increased proliferation rates due to loss of contact inhibition or tumor development in general.

Tyrphostin ErbB2 Inhibitors AG825 and AG879 Have Non-specific Suppressive Effects on gp130/ STAT3 Signaling

Although the interaction between gp130 and the ErbB family has frequently been shown in cancer cells, the mechanism of this interaction remains unclear and controversial. In the present study, we found that specific tyrphostin inhibitors of ErbB2 (AG825 and AG879), but not ErbB1 inhibitor (AG1478), suppressed IL-6-induced tyrosine phosphorylation of STAT3 in schwannoma cells. However, biochemical evidence for transactivation of ErbB2 by IL-6 was not observed. Additionally, the inhibition of ErbB2 expression, with either a specific RNAi or transfection of an ErbB2 mutant lacking the intracellular domain did not inhibit the IL-6-induced tyrosine phosphorylation of STAT3. Thus, it seems that tyrphostins, which are known as specific inhibitors of the ErbB2 kinase, may have non-specific suppressive effects on the IL-6/STAT3 pathway.

Identification of direct regulatory targets of the transcription factor Sox10 based on function and conservation

BackgroundSox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs.ResultsWe describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin) were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay.ConclusionIn sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.