Reduction of sporadic and neurofibromatosis type 2-associated vestibular schwannoma growth in vitro and in vivo after treatment with the c-Jun N-terminal kinase inhibitor AS602801.

OBJECTIVE: Vestibular schwannomas (VSs) are benign nerve sheath tumors that result from mutation in the tumor suppressor gene NF2, with functional loss of the protein merlin. The authors have previously shown that c-Jun N-terminal kinase (JNK) is constitutively active in human VS cells and plays a central role in their survival by suppressing accumulation of mitochondrial superoxides, implicating JNK inhibitors as a potential systemic treatment for VS. Thus, the authors hypothesized that the adenosine 5'-triphosphate-competitive JNK inhibitor AS602801 would demonstrate antitumor activity in multiple VS models. METHODS: Treatment with AS602801 was tested in primary human VS cultures, human VS xenografts, and a genetic mouse model of schwannoma (Postn-Cre;Nf2flox/flox). Primary human VS cell cultures were established from freshly obtained surgical tumor specimens; treatment group media was enriched with AS602801. VS xenograft tumors were established in male athymic nude mice from freshly collected human tumor. Four weeks postimplantation, a pretreatment MRI scan was obtained, followed by 65 days of AS602801 (n = 18) or vehicle control (n = 19) treatment. Posttreatment MRI scans were used to measure final tumor volume. Tumors were then harvested. Finally, Postn-Cre;Nf2flox/flox mice were treated with AS602801 (n = 10) or a vehicle (n = 13) for 65 days. Posttreatment auditory brainstem responses were obtained. Dorsal root ganglia from Postn-Cre;Nf2flox/flox mice were then harvested. In all models, schwannoma identity was confirmed with anti-S100 staining, cell proliferation was measured with the EdU assay, and cell death was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. All protocols were approved by the local institutional review board and Institutional Animal Care and Use Committees. RESULTS: Treatment with AS602801 decreased cell proliferation and increased apoptosis in primary human VS cultures. The systemic administration of AS602801 in mice with human VS xenografts reduced tumor volume and cell proliferation. Last, the AS602801-treated Postn-Cre;Nf2flox/flox mice demonstrated decreased cell proliferation in glial cells in the dorsal root ganglia. However, AS602801 did not significantly delay hearing loss in Postn-Cre;Nf2flox/flox mice up to 3 months posttreatment. CONCLUSIONS: The data suggest that JNK inhibition with AS602801 suppresses growth of sporadic and neurofibromatosis type 2-associated VSs. As such, AS602801 is a potential systemic therapy for VS and warrants further investigation.

Primary culture of human vestibular schwannomas.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

p75NTR is highly expressed in vestibular schwannomas and promotes cell survival by activating nuclear transcription factor κB.

Vestibular schwannomas (VSs) arise from Schwann cells (SCs) and result from the loss of function of merlin, the protein product of the NF2 tumor suppressor gene. In contrast to non-neoplastic SCs, VS cells survive long-term in the absence of axons. We find that p75(NTR) is overexpressed in VSs compared with normal nerves, both at the transcript and protein level, similar to the response of non-neoplastic SCs following axotomy. Despite elevated p75(NTR) expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75(NTR) . Furthermore, treatment with proNGF protects VS cells from apoptosis due to c-Jun N-terminal kinase (JNK) inhibition indicating that p75(NTR) promotes VS cell survival. Treatment of VS cells with proNGF activated NF-κB while inhibition of JNK with SP600125 or siRNA-mediated knockdown reduced NF-κB activity. Significantly, proNGF also activated NF-κB in cultures treated with JNK inhibitors. Thus, JNK activity appears to be required for basal levels of NF-κB activity but not for proNGF-induced NF-κB activity. To confirm that the increase in NF-κB activity contributes to the prosurvival effect of proNGF, we infected VS cultures with Ad.IκB.SerS32/36A virus, which inhibits NF-κB activation. Compared with control virus, Ad.IκB.SerS32/36A significantly increased apoptosis including in VS cells treated with proNGF. Thus, in contrast to non-neoplastic SCs, p75(NTR) signaling provides a prosurvival response in VS cells by activating NF-κB independent of JNK. Such differences may contribute to the ability of VS cells to survive long-term in the absence of axons.

Inhibition of c-Jun N-terminal kinase activity enhances vestibular schwannoma cell sensitivity to gamma irradiation.

BACKGROUND: Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS. OBJECTIVE: To determine the extent to which JNK signaling contributes to VS cell radiosensitivity. METHODS: Primary human VS cultures, derived from acutely resected tumors, received single doses (5-40 Gy) of gamma irradiation. Histone 2AX phosphorylation, a marker of IR-induced DNA damage, was assayed by Western blot and immunostaining. ROS levels were quantified by measuring 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. Cell apoptosis was determined by terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling. RESULTS: The JNK inhibitors SP6000125 and I-JIP reduced histone 2AX phosphorylation after IR. They also increased H2DCFDA fluorescence in nonirradiated cultures and significantly increased IR-induced (5-10 Gy) H2DCFDA fluorescence 72 hours, but not 2 hours, after IR. Finally, I-JIP (50 µmol/L) significantly increased VS cell apoptosis in cultures treated with 20 to 40 Gy. I-JIP (20 µmol/L), SP600125 (20 µmol/L), and JNK1/2 short interfering RNA knockdown each increased VS cell apoptosis in cultures treated with 30 to 40 Gy, but not lower doses, of IR. CONCLUSION: Inhibition of JNK signaling decreases histone 2AX phosphorylation and increases ROS and apoptosis in VS cells after gamma irradiation. These results raise the possibility of using JNK inhibitors to increase the effectiveness of radiosurgery for treatment of VSs.

Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons.

Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2'-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to promote rather than inhibit nerve fiber regeneration.

p75(NTR) expression and nuclear localization of p75(NTR) intracellular domain in spiral ganglion Schwann cells following deafness correlate with cell proliferation.

Spiral ganglion Schwann cells (SGSCs) myelinate spiral ganglion neurons (SGNs) and represent a potential source of neurotrophic support for SGNs. Deafening due to loss of hair cells results in gradual degeneration and death of SGNs. Successful efforts to maintain or regenerate a functional auditory nerve may depend on a healthy population of SGSCs, yet the responses of SGSCs to neural injury remain largely unknown. Here we investigate the role of p75(NTR) in SGSC responses to gradual denervation. Following deafening, SGSCs in the osseous spiral lamina (OSL) and, subsequently, in Rosenthal's canal (RC) expressed elevated p75(NTR) compared to hearing controls. p75(NTR)-positive cells co-labeled with S100 and RIP antibodies (Schwann cell markers), but not with anti-neurofilament. The pattern of p75(NTR) expression mirrored the pattern of neural degeneration, beginning in the OSL of the cochlea base and later extending into the apex. SGSCs expressed sortilin, a p75(NTR) co-receptor for pro-neurotrophins. Both pro-nerve growth factor (pro-NGF) and pro-brain derived neurotrophic factor (proBDNF) induced apoptosis in cultured SGSCs. Deafened animals exhibited significantly higher levels of SGSC proliferation (as measured by BrdU uptake) compared to hearing animals while total Schwann cell density remained stable, suggesting a tight regulation of SGSC proliferation and cell death. SGSCs undergoing cell division lose p75(NTR) expression from the cell surface and demonstrate nuclear localization of the intracellular domain (ICD), raising the possibility that p75(NTR) cleavage and ICD nuclear localization regulate SGSC proliferation. These results suggest that p75(NTR) contributes to SGSC responses to deafening and neural degeneration.

Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides.

Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase-signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.

Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth.

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl-2 prevents SGN death but inhibits neurite growth. Here we assessed the consequences of Bcl-2 targeted to either the mitochondria (GFP-Bcl-2-Maob) or the endoplasmic reticulum (ER, GFP-Bcl-2-Cb5) on cultured SGN survival and neurite growth. Transfection of wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob increased SGN survival, with GFP-Bcl-2-Cb5 providing the most robust response. Paradoxically, expression of GFP-Bcl-2-Maob results in SGN death in the presence of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears to be specific for neurotrophin signaling, insofar as coexpression of constitutively active mitogen-activated kinase kinase (MEKDeltaEE) or phosphatidyl inositol-3 kinase (P110), but not other prosurvival stimuli (e.g., membrane depolarization), also results in the loss of SGNs expressing GFP-Bcl-2-Maob. MEKDeltaEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT-3 or MEKDeltaEE. However, wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, and GFP-Bcl-2-Maob inhibit neurite growth even in the presence of neurotrophins, MEKDeltaEE, or P110. Historically, Bcl-2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl-2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl-2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. (c) 2010 Wiley-Liss, Inc.

The ErbB inhibitors trastuzumab and erlotinib inhibit growth of vestibular schwannoma xenografts in nude mice: a preliminary study.

OBJECTIVE: To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts. METHODS: Vestibular schwannoma xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor trastuzumab or saline for 2 weeks. Animals also received bromodeoxyuridine injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging during the treatment period. Cell death was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA. RESULTS: Tumors can be distinguished with T2-weighted magnetic resonance imaging sequences. Trastuzumab significantly reduced the proliferation of VS cells compared with control (p < 0.01) as analyzed by bromodeoxyuridine uptake. Control tumors demonstrated slight growth during the 12-week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p < 0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA-positive VS cells (p < 0.01). CONCLUSION: In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice, raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis Type 2.

Effects of ErbB2 signaling on the response of vestibular schwannoma cells to gamma-irradiation.

OBJECTIVE: 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 gamma-irradiation from a Cs gamma-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-1beta (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.

p75NTR and sortilin increase after facial nerve injury.

OBJECTIVES: After axotomy, Schwann cells (SCs), required for successful nerve regeneration, undergo a number of cellular changes including dedifferentiation, proliferation, expression of molecules that support axon growth, and apoptosis. This study investigated the role of p75, sortilin, and proneurotrophins in SC survival after facial nerve (FN) axotomy. STUDY DESIGN: Preliminary animal study. METHODS: With use of FN SCs, expression of p75 and its coreceptor sortilin were quantified by immunofluorescence on days 12, 22, and 52 after axotomy in vivo and by Western blot in vitro. Contralateral FNs served as a control. SC apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). To verify a causative role for p75 in FN SC death, cultured FN SCs were treated with pro-nerve growth factor (NGF), and apoptosis was determined by TUNEL. RESULTS: Expression of p75 and sortilin increased in FN SCs distal (P < .05) to the axotomy compared with the contralateral controls for all time points. SC apoptosis also significantly increased in the distal segment compared with the contralateral and proximal portions (P < .05). ProNGF, a p75 ligand, increased apoptosis and p75 expression in primary FN SC cultures. CONCLUSION: FN axotomy increases p75 and sortilin expression in SCs, which correlates with increased apoptosis. These findings suggest roles for p75 and sortilin in SC loss after FN injury. Sortilin is a novel target in promoting FN healing after injury.

Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout.

We investigated whether there are compensatory changes in the coronary microvasculature, cardiac lipid metabolism, and myocyte ultrastructure associated with ventricular enlargement in male rainbow trout. Epicardial tissue was sampled at different stages of sexual maturation, and we estimated arterial capillary density, intercapillary diffusion distance, and applied a diffusion model to predict PO(2) at different workloads. We also measured biochemical indices of lipid metabolism and estimated fractional volumes of mitochondria and myofibrils in myocytes. Immature fish with nonenlarged ventricles had the highest capillary length densities (1620+/-158 mm mm(-3)). Maturing trout with moderate ventricular hypertrophy had lower capillary length densities (1103+/-58 mm mm(-3)) and similar diffusion distances (13.9+/-0.7 microm) compared with immature fish (11.7+/-0.9 microm). The largest ventricles had intermediate capillary length densities (1457+/-288 mm mm(-3)) and diffusion distances (12.8+/-0.8 microm). Modelling predicted that enlarged ventricles would not become anoxic even at maximal workloads. Biochemical markers of fatty acid metabolism and aerobic capacity were unchanged with hypertrophy. Volume densities of mitochondria and myofibrils were also not influenced by cardiac growth. In summary, ventricle hypertrophy results in expansion of the coronary capillary bed and the maintenance of the epicardial capacities for fat and oxidative metabolism.