|Home | About | Journals | Submit | Contact Us | Français|
Vestibular schwannomas (VS) frequently express high levels of activated AKT. Small-molecule inhibitors of AKT signaling 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 hours 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 nine 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.
Vestibular schwannomas (VS) are histologically benign nerve sheath tumours that originate on the superior or inferior vestibular branches of cranial nerve VIII.1 Clinical symptoms of VS include sensorineural hearing loss, balance abnormalities, tinnitus, vertigo, facial weakness, hydrocephalus, blindness, and even death. VS can be divided into four general categories: unilateral sporadic VS, neurofibromatosis type 2 (NF2)-associated VS, cystic schwannomas, and malignant schwannomas. Among VS, unilateral sporadic tumours are by far the most common. The development of bilateral VS is the hallmark of NF2, an autosomal dominant disease caused by mutations in the Neurofibromatosis 2 (NF2) tumour suppressor gene, which encodes a tumour suppressor protein named merlin, for moesin, ezrin, and radixin-like protein.2,3 Cystic schwannomas are a particularly aggressive group of unilateral schwannomas that often grow rapidly. Mutations in the NF2 gene have also been frequently found in sporadic VS and cystic schwannomas.1,4 Malignant VS, also called triton tumours, are rare but aggressive variants of VS. They occur either sporadically or following radiotherapy and are uniformly fatal.5,6
Currently, no known medical therapies are available for the treatment of VS. While improved diagnostic techniques, such as magnetic resonance imaging (MRI), detect these tumours at early stages, surgical excision or stereotactic radiation remain the only viable treatment options. Unfortunately, complications from treatment, such as hearing loss, facial weakness, dizziness, intracranial bleeding, and stroke, remain major concerns.1 Furthermore, radiation treatment may cause malignant transformation and/or growth acceleration of schwannomas.7–9 The current management of malignant schwannomas is gross surgical resection followed by adjuvant radiotherapy. However, due to the location and extent, total excision of these invasive tumours is difficult to achieve.10 Currently available chemotherapy regimens have little efficacy in treating triton tumours.11,12 Together, these factors underscore the importance of developing effective chemotherapeutic agents that stop tumour growth or completely eradicate VS and malignant scwhannomas.
A better understanding of the mechanisms underlying VS tumourigenesis may provide the tools necessary to design novel targeted therapies. The loss of functional merlin protein has been implicated in VS development. Recent studies suggest that merlin plays important roles in regulating actin cytoskeleton-mediated processes, adherens junction formation, and cell proliferation;13 however, the tumour suppressor role of merlin remains elusive. We and others have identified several signaling pathways frequently deregulated in VS, including the PI3K/AKT pathway.4,14–19 In addition to slightly elevated AKT mRNA and protein levels, VS tumours express substantially higher levels of phosphorylated, activated AKT than paired vestibular nerve specimens. Since the PI3K/AKT pathway serves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes and responses, such as cell survival, cell proliferation, insulin response, stress response, and differentiation,20 its activation likely contributes to tumourigenesis. Thus, the PI3K/AKT pathway is an attractive therapeutic target for VS, and small-molecule inhibitors of AKT signaling may have therapeutic potential in suppressing the growth of these tumours.
OSU-03012 is a recently licensed (AR-12; Arno Therapeutics, Inc.) novel derivative of the cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex™), but devoid of COX-2 inhibitory activity. It potently induces apoptosis in several types of cancer cells21–25 and is currently undergoing preclinical development in the Rapid Access to Intervention Development (RAID) program at NCI. The mechanism by which OSU-03012 induces apoptosis is mediated, at least in part, through inhibition of phosphoinositide-dependent kinase 1 (PDK-1), an upstream kinase that phosphorylates AKT. Since VS frequently exhibit activation of AKT, we evaluated the growth inhibitory and anti-tumour activities of OSU-03012 on cultured human and mouse Schwann cells and schwannoma cells, in addition to schwannoma xenografts in several combined immunodeficiency (SCID) mice. Our results demonstrated that OSU-03012 effectively inhibited cell proliferation and induced apoptosis in both benign and malignant human schwannoma cells, as well as mouse Nf2−/− Schwann cells and Nf2−/− schwannoma cells, with IC50 values in the low micro-molar range. We also showed that OSU-03012 possessed potent anti-tumour activity in malignant schwannoma xenografts in SCID mice.
OSU-03012 was synthesized as previously described21 with purities exceeding 99% as determined by nuclear magnetic resonance spectroscopy (300 mhZ).
The Ohio State University Institutional Review Board approved Human Subjects Protocols for the acquisition of surgically removed VS specimens from patients and normal nerves from organ donors through Lifeline of Ohio. VS were resected with informed patient consents, and their histological type was confirmed by a neuropathologist. Vestibular schwannoma and normal nerve tissues were placed in Dulbecco’s-modified Eagle (DME) medium (Invitrogen) and brought to the research laboratory shortly after resection.
To prepare VS cell cultures, tumour tissues were cut into small (< 1 mm) pieces and incubated in DME medium containing 10% fetal bovine serum (FBS), 130 U/ml collagenase type I, 0.6 U/ml dispase, 100 I.U./ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen), for six hours at 37°C with moderate shaking. Dissociated tissues were further triturated by passing through a narrow Pasteur pipette. Cell suspension was collected by centrifugation, resuspended in DME medium supplemented with 10% FBS, 10 nM recombinant human NRG1-β1/HRG1-β1 EGF domain (heregulin; R&D Systems), 2μM forskolin (Sigma-Aldrich), 100 I.U./ml penicillin, and 100 μg/ml streptomycin, and plated on dishes coated with poly-L-lysine and laminin (Sigma-Aldrich).
For preparing human Schwann cell cultures, femoral nerves from organ donors were cut into small pieces and incubated at 37°C in DME medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 0.05 mg/ml streptomycin for 7 days for in vitro Wallerian degeneration.26 This pretreatment medium was replaced every two days. Pretreated nerve tissues were dissociated, and cell suspension was plated in medium containing heregulin and forskolin as described above for VS cells. Using a similar method, normal mouse Schwann cells were prepared from sciatic nerves harvested from FVB/N mice at four to eight weeks old.
Mouse Nf2−/− Schwann cells were prepared by two methods. Schwann cells derived from sciatic nerves of Nf2flox2/flox2 mice27 were infected with AdCMVCre recombinant adenoviruses carrying a cytomegalovirus (CMV) promoter-driven Cre gene (Gene Transfer Vector Core at University of Iowa, Iowa City, IA) to delete the Nf2 gene. Alternatively, sciatic nerves from Nestin-CreER;Nf2flox2/flox2 mice (E.M. Akhmametyeva, L.-S. Chang, and colleagues, Nationwide Children’s Hospital and The Ohio State University) were used to prepare Schwann cell cultures, followed by treatment with tamoxifen for 48 hrs.28 To detect the deletion of the Nf2 gene, genomic DNA was isolated from a portion of cells using the Gentra Puregene Cell Kit (Qiagen) and analysed by the polymerase chain reaction technique accordingly27 We found that the Nf2 gene was deleted in at least 90% of the Schwann cells obtained by either method.
In addition, we prepared mouse Nf2−/− schwannoma cells from the tumour developed in the P0Cre;Nf2flox/flox mice with conditional Nf2 inactivation in Schwann cells.27 For comparison, we established mouse NF2P2.4-T schwannoma cells from a schwannoma developed in a transgenic mouse carrying a 2.4-kb NF2 promoter-driven SV40 T-antigen oncogene (E.M. Akhmametyeva, L.-S. Chang, and colleagues, Nationwide Children’s Hospital and The Ohio State University).29 The human malignant schwannoma HMS-97 cells have been described previously.30 Both mouse NF2P2.4-T and human HMS-97 schwannoma cells were grown in DME medium supplemented with 10% FBS.
VS and HMS-97 cells were grown in polylysine/laminin-coated 96-well plates (5 × 103 to 1 × 104 per well). After incubation at 37°C for 24 hrs, cells were treated with various concentrations of OSU-03012 or DMSO as a vehicle control at 37°C for another 48 hrs. To assess cell proliferation, the CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega) was used according to the manufacturer’s instructions. Briefly, 20μl of CellTiter 96® AQueous One Solution Reagent were added to each well and incubated at 37°C for 1 to 4 hrs. The amount of colored formazan produced in the cells was measured at 490nm using a Victor3 1420 Multilabel Counter (Perkin Elmer). The percentage of cell proliferation was plotted against the concentrations of OSU-03012, and the IC50 (defined as the drug concentration at which the population of viable cells was reduced by 50%) was determined using CalcuSyn software (Biosoft). The experiments were replicated six times, and the mean IC50 was calculated.
VS and HMS-97 cells were treated with increasing concentrations of OSU-03012 or 0.05% DMSO as a negative control for 8 to 24 hrs. Treated cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentrations of clear lysates were determined using the Bio-Rad Protein Dye Reagent. Equal amount (20μg/each) of protein was electrophoresed on an 8% SDS-polyacrylamide gel, followed by electroblotting to an Immobilon-P membrane (Millipore). After protein transfer, the membrane was blocked with 5% (w/v) non-fat dry milk in 10 mM Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature for 1 hr and then incubated with a primary antibody (1:1000 dilution) at 4°C overnight. After washing with TBST three times, a horseradish peroxidase-conjugated secondary antibody (1:2000 dilution) was added to the membrane at room temperature for 1 hour, followed by incubation with the ECL Plus™ Western Blotting Reagent (GE Healthcare). The chemiluminescent activity was captured by exposure to Fiji medical X-ray films or scanning with a Typhoon 9400 phosphoimager. Primary antibodies used include: anti-AKT, anti-phospho-AKT(Ser473), anti-phospho-AKT(Thr308), anti-phospho-GSK-3β (Ser 9), and anti-caspase 9 (all from Cell Signaling Technology). Anti-α-tubulin and anti-β-actin antibodies (Rockland Immunochemicals) were used as loading controls.
Apoptosis was detected using the DeadEnd Fluorometric TUNEL system (Promega). VS and HMS-97 cells grown on polylysine/laminin-coated coverslips (BD Biosciences) were treated with 7.5 μM of OSU-03012 or 0.05% DMSO as the control for various durations. Treated cells were fixed in 4% paraformaldehyde for 25 minutes, permeablized in 0.2% Triton X-100 in PBS for 5 minutes at room temperature, and then labeled with fluorescein-12-dUTP using recombinant terminal deoxynucleotidyl transferase. Labeled cells were washed three times in PBS and covered with a drop of ProLong Gold Antifade Reagent with DAPI (Invitrogen) prior to mounting onto a glass slide. Under a Leica DM4000 fluorescence microscope, DAPI stained all nuclei in blue and fluorescein-12-dUTP incorporation resulted in localized green fluorescence within the nucleus of apoptotic cells.
Apoptosis was also confirmed by the appearance of the active large fragment of cleaved caspase 9. HMS-97 and VS cells were exposed to different concentrations of OSU-03012 for 48 hrs. Soluble cell lysates were prepared and used to detect pro- and cleaved-caspase 9 by Western blot using an anti-caspase 9 antibody (catalog No. 9502; Cell Signaling Technology) as described above.
Schwannoma xenografts were established in severe combined immunodeficiency mice (SCID) and MRI scans were conducted as previously described.30 Briefly, human malignant schwannoma HMS-97 cells (2.5 × 105 cells/mouse) were washed in PBS and suspended in 0.2 ml of Matrigel® (1:1 diluted in PBS; BD Biosciences). The right flank of SCID mice was shaved and prepped using aseptic technique. An 18-gauge needle was used to inject schwannoma cells into the subcuticular plane raising a small wheal in the region overlying the sciatic nerve. The injected mice were observed for one week and were then divided into two groups. One group of mice was administered 200 mg/kg/day of OSU-03012 by oral gavage. The other group of mice served as controls and were gavage-fed a similar volume of the vehicle, consisting of methylcellulose (0.5%, w/v) and Tween 80 (0.1%, w/v) in sterile water. After nine weeks of treatment, mice were examined under anesthesia using a 4.7 Tesla/cm magnetic resonance imaging (MRI) scanner (Bruker) as described previously.30 T1-weighted axial and coronal images were obtained using a spin echo sequence with TR of 550–600 ms and TE of 10.5 ms. In-plane resolution was 156 μm on the axial and 195 μm on the coronal images, and the slice thickness was 0.8 mm with a 0.2-mm gap between slices. Multi-planar tumour volumes were determined by manually tracing tumour areas on axial and coronal images, followed by adding the traced areas from all slices depicting the tumour and multiplying with the distance between slices.
Xenograft tumours grown in SCID mice with or without OSU-03012 treatment were dissected, fixed in 10% buffered formalin, and embedded in paraffin. Five-micron tissue sections were obtained and processed for standard hematoxylin-eosin staining or immunostaining with antibodies against phospho-AKT.19 Negative controls were treated with the same immunostaining procedure except without the primary antibody.
Since the PI3K/AKT pathway is frequently activated in human VS, we tested whether OSU-03012 could effectively inhibit the growth of benign VS and malignant schwannoma HMS-97 cells, compared with normal human Schwann cells in culture. Exposure of primary human VS cells to OSU-03012 resulted in a dose-dependent inhibition of cell proliferation (Figures 1A and 1B). To exclude the possibility of individual differences in drug sensitivity, we exposed six VS cultures from different patients with various concentrations of OSU-03012 and determined the IC50 values. In addition, as heregulin has been shown to synergize with intracellular cAMP to enhance Schwann cell proliferation,31 we performed OSU-03012 treatment in the presence or absence of heregulin and forskolin. The IC50 values for various preparations of human VS cells tested in the absence of heregulin ranged from 2.1 to 4.0μM (Table 1). In the presence of heregulin, only a slight increase in the IC50 values for various VS cell cultures was observed (IC50 ranging from 2.7 to 5.4 μM). Similarly, a dose-dependent inhibition of cell proliferation by OSU-03012 was observed for human malignant schwannoma HMS-97 cells with an average IC50 value of 2.6μM (Figure 1C and Table 1). For comparison, we also prepared primary normal human Schwann cells and treated them with various concentrations of OSU-03012. Normal human Schwann cells were more resistant to growth inhibition by OSU-03012 (Figure 1D). The IC50 values for various preparations of normal human Schwann cells cultured in the presence of heregulin were greater than 12 μM. These results indicate that OSU-03012 potently inhibits the growth of both benign human VS and malignant schwannoma cells in culture.
Since most, if not all, VS harbor mutations in the NF2 gene1–3 and since mouse Nf2−/− schwannoma cells also frequently exhibit up-regulation of the PI3K/Akt pathway,16 we compared the growth inhibitory effect of OSU-03012 on various mouse Nf2+/+ or Nf2−/− Schwann cells and schwannoma cells. Mouse Nf2−/− schwannoma cells derived from a spontaneously occurring schwannoma in the P0Cre;Nf2flox/flox mouse27 showed a dose-dependent growth inhibition when exposed to various concentrations of OSU-03012 (Figure 2A). Similarly, exposure of primary mouse Nf2−/− Schwann cells to OSU-03012 resulted in a dose-dependent inhibition of cell proliferation (Figure 2B). The IC50 value for mouse Nf2−/− schwannoma cells was about 2.4 μM, while that for mouse Nf2−/− Schwann cells was about 2.9μM (Table 1). In contrast, wild-type mouse Schwann cells were substantially less sensitive to growth inhibition by OSU-03012 (Figure 2C) with an IC50 value of greater than 20μM (Table 1). As an additional control, we used mouse schwannoma cells derived from a schwannoma generated in a transgenic NF2P2.4-T mouse carrying the SV40 T-antigen gene driven under the control of the 2.4-kb NF2 promoter. These NF2P2.4-T schwannoma cells express the viral T-antigen oncoprotein but carry the wild-type Nf2 gene. Interestingly, these NF2P2.4-T schwannoma cells were also relatively insensitive to growth inhibition by OSU-03012, compared with mouse Nf2−/− schwannoma and Nf2−/− Schwann cells (compare Figure 2D to Figures 2A and 2B). The IC50 value for these NF2P2.4-T schwannoma cells was greater than 12μM (Table 1). Together, these results imply that loss of a functional Nf2 gene correlates with the higher sensitivity of mouse Schwann cells and schwannoma cells to growth inhibition by OSU-03012.
One major biochemical action of OSU-03012 is the inhibition of PDK-1, which phosphorylates and activates AKT.21 To examine the phosphorylation status of AKT in HMS-97 and VS cells treated with various concentrations of OSU-03012, Western blots using antibodies specific to phospho-AKT at the threonine-308 [p-AKT(Thr308)] and serine-473 [p-AKT(Ser473)] sites, and total AKT were performed. A dose-dependent inhibition of AKT phosphorylation at the threonine-308 site, which is the primary phosphorylation site of PDK1, was observed in both HMS-97 (Figure 3A) and VS cells (Figure 3B). The inhibition of Akt phosphorylation at the threonine-308 site in OSU-03012-treated HMS-97 cells appeared to be less than that in OSU-03012-treated VS cells; however, the reason for this observation is presently not known. Similarly, we detected a dose-dependent inhibition of AKT phosphorylation at the serine-473 site when cells were treated with increasing concentrations of OSU-03012. Likewise, phosphorylation of glycogen synthase kinase-3β [p-GSK-3β (Ser9)], a downstream substrate of AKT, was also inhibited by OSU-03012 in a dose-dependent manner. In contrast, the expression level of total AKT remained constant in both the malignant and benign schwannoma cells treated with up to 7.5 μM of OSU-03012. Furthermore, we showed that under similar treatment conditions, OSU-03012 had little or no effect on Akt phosphorylation in both NF2P2.4-T schwannoma cells (Figure 3C) and normal mouse Schwann cells (Figure 3D), both of which were relatively insensitive to growth inhibition by OSU-03012 (Figure 2). These results indicate that the inhibition of AKT phosphorylation at both the threonine-308 and serine-473 sites by OSU-03012 correlates with its anti-proliferative effect in both human malignant and benign schwannoma cells.
Since AKT is critical to cell proliferation and cell survival, we next examined whether OSU-03012 induced programmed cell death in both HMS-97 and VS cells by two methods, TUNEL and cleavage of caspase 9. For the TUNEL assay, we treated HMS-97 and VS cells with 7.5 μM of OSU-03012 for various durations before end-labelling to detect apoptosis-induced DNA fragmentation. TUNEL-positive HMS-97 cells were found as early as 6 hr after treatment (data not shown), and were readily observed after 14 hrs (Figure 4A). Nearly all HMS-97 cells were TUNEL-labeled 24 hrs after OSU-03012 treatment, while DMSO-treated control cells showed no labelling. Similarly, TUNEL-positive VS cells were detected 18 hrs after OSU-03012 treatment, and nearly all OSU-03012-treated cells were labeled after 24 hrs (Figure 4B). To confirm apoptotic induction in OSU-03012-treated cells, Western blot analysis to detect cleaved caspase 9 was performed. Treatment with increasing concentrations of OSU-03012 resulted in a dose-dependent decrease of caspase 9 with a concomitant increase of the 35-kDa active fragment of cleaved caspase 9, evidence of apoptotic induction, in both HMS-97 and VS cells (Figure 5). In contrast, OSU-03012 treatment had little or no effect on caspase 9 cleavage in normal mouse Schwann cells under similar conditions (data not shown). Together, these results indicate that OSU-03012 potently induced apoptosis in both benign and malignant schwannoma cells.
To evaluate the anti-tumour potential of OSU-03012, SCID mice bearing HMS-97 malignant schwannoma xenografts were treated orally with OSU-03012 at 200 mg/kg/day or with vehicle for nine weeks. Then, the tumour volume of schwannoma xenograft in each mouse was quantified using high-field small-animal MRI.30 Treatment of mice with OSU-03012 inhibited HMS-97 tumour growth by 55%, relative to the vehicle-treated control (Figure 6). This in vivo efficacy after oral administration confirmed the oral bioavailability of OSU-03012, which was previously determined to be approximately 40% in rats using the same vehicle.32 SCID mice appeared to tolerate OSU-03012 at 200 mg/kg/day for nine weeks. We did not observe any signs of toxicity or significant changes in body weight of the treated animals, compared with the vehicle-treated group. Importantly, histopathological examination revealed that the malignant schwannoma xenograft tumour from the OSU-03012-treated mouse harbored islands of necrosis, while those from the vehicle-treated control exhibited minimal necrosis (Figure 7A). To correlate this in vivo tumour-suppressive response to an intratumoural biomarker of drug activity, HMS-97 xenograft tumours from untreated and treated mice were harvested, and tumour sections were immunostained with the anti-p-AKT(Ser473) antibody. As shown in Figure 7B, the phosphorylated AKT protein was readily detected in both the nucleus and cytoplasm of HMS-97 tumour cells treated with the vehicle control. In contrast, the level of phospho-AKT staining was substantially reduced in OSU-03012-treated tumour cells (Figure 7B). Collectively, these results demonstrate that OSU-03012 possessed potent anti-tumour activity in malignant schwannoma xenografts in SCID mice.
Vestibular schwannomas (VS) are intracranial tumours arising from Schwann cells of cranial nerve VIII. VS are frequently seen in patients with NF2 and some of them have multiple schwannomas, which may be difficult to remove.1 Currently, no medical therapies are available for the treatement of all types of benign VS. Malignant schwannomas are rare but highly aggressive with a 5-year survival rate of only 10–20%.6 These tumours do not respond to currently available chemotherapy, and total excision is difficult to achieve due to their location and extent at presentation.10–12 Therefore, the development of chemotherapies to stop tumour growth or completely eradicate VS and malignant schwannomas is of urgent clinical need. Since both VS17,19 and malignant schwannomas (Figures 3 and and7)7) frequently express high levels of activated AKT, drugs targeting the AKT pathway, such as OSU-03012, are likely to have therapeutic potential in suppressing the growth of these tumours. In the present study, we assessed the in vitro and in vivo efficacy of OSU-03012 in human benign VS and malignant schwannoma HMS-97 cells. We showed that OSU-03012 possesses potent growth inhibitory and anti-tumour activities on both benign and malignant human schwannoma cells, as well as mouse Nf2−/− Schwann cells and schwannoma cells.
Consistent with previous studies using other human cancer cells,21–24 OSU-03012 potently induces growth inhibition and apoptosis in both benign and malignant schwannoma cells in association with PDK-1 inhibition and subsequent inactivation of Akt and downstream signaling molecules. Intriguingly, we found a dose-dependent reduction of AKT phosphorylation at both the threonine-308 and serine-473 sites in HMS and VS cells upon treatment with increasing concentrations of OSU-03012. Although the threonine-308 site is considered the primary phosphorylation site by PDK1, three models have been proposed for AKT phosphorylation at the serine-473 site, including phosphorylation by PDK1, by a putative kinase named PDK2, or by autophosphorylation.33 If the serine-473 site is phosphorylated by a kinase other than PDK1,34 our results argue that OSU-03012 may also inhibit the other AKT kinase, PDK2. In addition, OSU-03012 may induce apoptosis in cancer cells by affecting pathways other than PDK-1/Akt signaling, including the disruption of mitochondrial membrane potential and activation of caspase-9,23,35 induction of endoplasmic reticulum stress responses,36 inhibition of p21-activated kinase 1 (PAK1) activity,24 inhibition of Janus-activated kinase 2/signaling transducer activator of transcription 3 and mitogen-activated protein kinase (MAPK) pathways, and down-regulation of cyclins A and B and the inhibitor of apoptosis protein members, X-linked inhibitor of apoptosis, and survivin.25 Consistently, we also found that OSU-03012 treatment led to activation of caspase 9 in both VS and HMS-97 malignant schwannoma cells.
VS frequently harbor mutations in the NF2 tumour suppressor gene.1–3 Evidence suggests that merlin, the NF2 gene product, may exert its growth suppressive activity by inhibiting the PI3K/AKT pathway.18,37 PI3K is a membrane-associated lipid kinase that converts phosphatidylinositol diphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3), which recruits AKT from the cytoplasm to the cell membrane where it is phosphorylated and activated by PDK-1.20 Merlin could bind to the PI3K enhancer long isoform (PIKE-L), a brain-specific GTPase that binds to PI3K and stimulates its lipid kinase activity,37 and could inhibit PI3K activity by disrupting the binding of PIKE-L to PI3K. In addition, recent evidence suggests that AKT could also bind to and phosphorylate merlin on residues threonine-230 and serine-315, which abolish merlin’s intra-molecular interactions and binding to PIKE-L and other binding partners.18 Although the reciprocal experiment for the effect of merlin binding on AKT activity has not been demonstrated, it is conceivable that merlin inactivation, as frequently seen in VS, could result in activation of the PI3K/AKT pathway. Consistent with this notion, mouse Nf2−/− Schwann cells or schwannoma cells derived from mice with conditional Nf2 knockout in Schwann cells show activation of Akt.16 Analogous to human VS and malignant schwannoma cells, both mouse Nf2−/− Schwann cells and Nf2−/− schwannoma cells display higher sensitivity to inhibition of AKT by OSU-03012 than normal mouse Schwann cells and NF2P2.4-T mouse schwannoma cells. These results indicate that biallelic loss of a functional NF2 gene and activation of the AKT pathway in Nf2−/− schwannoma and Schwann cells correlate with chemotherapeutic sensitivity to OSU-03012.
It should be mentioned that the SV40 T-antigen oncoprotein is capable of transforming a variety of cell types. T antigen transforms cells by binding and inactivating the p53 and retinoblastoma protein family of tumour suppressors.38 The fact that the NF2P2.4-T mouse schwannoma cells are relatively resistant to growth inhibition by OSU-03012 compared with Nf2−/− mouse schwannoma or Schwann cells implies that T antigen induces tumour formation by targeting pathways other than the PI3K/AKT pathway. In addition, these results further implicate the specificity of OSU-03012 action to the AKT pathway. The NF2P2.4-T mouse schwannoma cells should be useful as negative controls for future testing of pathway-specific inhibitors.
Like many other key regulatory proteins, merlin is regulated by phosphorylation,39 possibly mediated through Rac1 and other Rho GTPase family members.40 Among the Rac/Cdc42 effectors, PAK1 and PAK2 can phosphorylate merlin, affecting merlin’s localization and function.37,39,41–43 Conversely, merlin inhibits PAK1, and the PAK1-specific inhibitors could reduce the growth of NF2-deficient mesothelioma cells.44 Interestingly, Porchia et al.24 recently demonstrated that OSU-03012 could also inhibit PAK activity in thyroid carcinoma cells. Since PAK expression is frequently up-regulated in NF2-deficient cells,4,16,42 experiments are in progress to examine whether OSU-03012 could inhibit PAK activation in schwannoma cells.
Two lines of evidence support the notion that OSU-03012 induces apoptosis in schwannoma cells. First, OSU-03012 treatment induced chromosome fragmentation in the nucleus of both VS and HMS-97 schwannoma cells as visualized by TUNEL staining. This effect is accompanied by the decreased level of activated AKT. Second, OSU-03012 treatment results in a dose-dependent cleavage of caspase 9, leading to the appearance of the 35-kDa active fragment of cleaved caspase 9. Cardone et al.45 previously showed that murine Akt could phosphorylate caspase on serine-196 and inhibit its protease activity. A phosphorylation-inactive caspase 9 mutant, when expressed in the cell, resulted in Akt-resistant induction of apoptosis. Thus, it is possible that inhibition of AKT phosphorylation and activation by OSU-03012 may allow cleavage and generation of an active caspase 9, ultimately leading to apoptosis. In this manner, OSU-03012 induces the caspase-dependent pathway of apoptosis in both benign VS and malignant HMS-97 schwannoma cells. It is noteworthy that OSU-03012 may also induce apoptosis via caspase-independent mechanisms in other tumour types.22,25,36
Chemotherapeutic agents that could reduce the size of VS while sparing associated nerves are attractive options for treating these tumours. OSU-03012 is a derivative of celecoxib, but lacks the COX-2 inhibitory activity of the parent drug.21 Such a unique feature may limit the known side effects of COX-2 inhibitors and make OSU-03012 an attractive choice for the treatment of benign tumours such as VS. Our in vivo data showing the effect of OSU-03012 on the growth of malignant schwannoma xenografts in SCID mice further corroborate the in vitro findings. Also, the results indicate the oral bioavailability and tolerability of OSU-03012 at the dose used in the mice, consistent with previous reports using murine xenograft models for liver, breast and lung cancers.46–48 Intriguingly, we observed that, in addition to reducing tumour size, OSU-03012 treatment induced necrosis within the HMS-97 malignant schwannoma xenograft grown in SCID mice. Although the mechanism underlying the induction of this necrosis is presently unknown, it is tempting to speculate that OSU-03012 may induce cell death by down-regulating genes that encode autocrine growth factors, cytokines, and/or angiogenic factors. Some of these genes could be downstream targets of the PI3K/AKT pathway. Consistent with this notion, we observed that the in vivo tumour-suppressive response to OSU-03012 activity correlates with the decrease of activated AKT in the xenograft tumour. Presently, we are conducting a similar experiment to test the in vivo efficacy of OSU-03012 using VS xenografts.
Several additional characteristics of OSU-03012 make it an attractive candidate for further drug development. Due to their intracranial location, treatment of VS may require drugs that penetrate the blood-brain barrier. Our preliminary analysis reveals that OSU-03012 effectively crosses the blood-brain barrier after intravenous dosing. OSU-03012 has also been reported to interact synergistically with other chemotherapeutic agents, such as the protein tyrosine kinase inhibitor imatinib mesylate49 and EGFR inhibitors,48 to suppress tumour growth. In addition, OSU-03012 could sensitize tumour cells to radiation.36 Pretreatment with OSU-03012 may lower stereotactic radiation doses to levels that minimize toxicity, preserve cranial nerve function, and decrease long-term risk for malignant transformation. Together, these results suggest that OSU-03012 has potential to become a novel treatment option for VS and malignant schwannomas.
We sincerely thank Georgeta Mihai for volumetric MRI analysis, Lifeline of Ohio for nerve specimens, Chang lab members for discussion and suggestions throughout the study, and Sarah S. Burns for critical reading of the manuscript. This work was supported by grants from the National Institute of Deafness and Communicative Disorders (DBW/LSC), the Department of Defense Neurofibromatosis Research Program (LSC), Children’s Tumor Foundation (AJ), The Triological Society (AJ/LSC/DBW), and the National Cancer Institute-supported Ohio State University Comprehensive Cancer Center.
Conflict of interest statement
All authors do not have any disclosure of potential conflict of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.