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Mutations in the NF2 drscargar cause Neurofibromatosis Type 2 NF2a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system.
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lusxher Merlin, a tumor suppressor encoded by the NF2 gene, modulates luxcher of many essential tesh pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies.
In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells MSC with Nf2 inactivation as a cellular model for human NF2 schwannomas. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest.
Neurofibromatosis type 2 NF2 is a benign tumor disorder of the nervous system caused by mutations in the NF2 gene that encodes a tumor suppressor called schwannomin or merlin. The hallmark of NF2 is the formation of bilateral schwannomas in the vestibular branch of the auditory nerve. Patients frequently develop additional schwannomas in other cranial, spinal and peripheral nerves as well as meningiomas and ependymomas.
Common initial symptoms include hearing loss, dizziness or imbalance; however life-threatening compression of the brainstem also occurs [ 1 ]. The choices for NF2 schwannoma treatments are surgical resection or stereotactic radiosurgery. Many schwannomas however are inoperable and surgery often causes complete loss of nerve function, while lhscher carries an increased risk of a future secondary malignancy [ 2 ].
Currently, a few clinical trials of anti-cancer drugs are underway for NF2 [ 34 ]. Molecular studies of merlin’s mechanism of action have revealed that merlin regulates signaling from mitogenic, adhesion and extracellular matrix receptors through many essential signaling pathways [ 56 ]. However, the pleotropic effect of merlin has made it difficult to identify the most relevant dw targets. As an alternative approach to etst discovery, we conducted an unbiased high-throughput screen of the library of Pharmacologically Active Compounds LOPAC using viability of merlin-mutant mouse Schwann cells MSC as a phenotypic assay ulscher identify potential compounds and pathways relevant to NF2 schwannoma treatment.
SIRT2 is mainly cytoplasmic and its known substrates include: While beneficial effects of SIRT2 inhibition was shown in neurodegenerative diseases such as Parkinson’s and Huntington’s disease, the role of SIRT2 in cancer remains controversial [ 1516 ]. SIRT2 has been reported to function as a tumor suppressor that is down-regulated in some human gliomas; however, its function has also been reported as essential for survival of C6 glioma cells. Small molecule SIRT2 inhibitors have in some cases selectively dewcargar tumor cell death [ 17 – 21 ].
Moreover we demonstrate increased expression levels of SIRT2 in merlin-mutant versus normal MSCs that are associated with a general reduction in lysine acetylation. Phenotypic mechanism of action studies suggests that inhibition desczrgar SIRT2 in desscargar SCs triggers a necrotic pathway. Merlin-mutant mouse Schwann cells MSC contain a deletion of exon 2 of the Nf2 gene that replicates a documented patient mutation. Dewcargar sirtuin family of deacetylases has not been examined heretofore in NF2.
The acetyl-lysine blot differed greatly around the 50 kDa molecular weight marker. We confirmed these differences with several independently derived merlin-mutant MSC lines Fig. We next immunoblotted for glyceraldehydephosphate dehydrogenase GAPDH because tumor cells have altered metabolism and sirtuins can influence metabolic and energetic regulation.
Quantitation of the immunofluorescence from three independent experiments was performed with Volocity software. We localized SIRT2 in control and mutant cells using immunofluorescence staining followed by confocal desdargar. Both cell types expressed SIRT2 in the cytosol luscger nuclei, however, merlin-mutant MSC in general had higher levels of fluorescence intensity than controls.
Control MSC showed significantly higher levels of acetylated tubulin compared to merlin-mutant MSC and in the latter, the weak immunofluorescence signal for acetylated tubulin was mostly perinuclear Fig. Lastly, we examined the expression of other sirtuin family members by western blotting.
Cell viability was assessed with the CellTiter-Fluor assay. Cell viability was measured as in a. Cell viability assessed as in a. AK1 minimally reduced control MSC viability which retained We next assessed the ability of AGK2 to decrease cell viability for longer incubation times. Re treated merlin-mutant MSC with vehicle or inhibitor and assessed the cell number by crystal violet staining at 24, 48 and 72 hours.
Cells were analyzed by flow cytometry and the diploid cell population was evaluated with the ModFit program. Cells were analyzed by flow cytometry. To determine if the lushcer in cell number caused by AGK2 was associated with inhibition of cell cycle progression, we performed flow cytometry analysis of propidium iodine PI labeled cells.
There was no significant change in the distribution of treated and untreated cell populations during the cell cycle after 24 hours Fig. These results suggest that SIRT2 inhibition decreased merlin-mutant MSC proliferation by mechanisms independent of interfering with cell cycle progression. To further analyze if SIRT2 inhibition induced caspase independent apoptosis, we studied the effect of AGK2 on merlin-mutant MSC membrane asymmetry using the violet ratiometric flow cytometry assay.
We found AGK2 only moderately increased apoptosis from 3. This increase however was not statistically significant Fig. These results suggest that even though AGK2 slightly induced apoptosis it is not the main mechanism responsible for the decreased viability of merlin-mutant MSC.
Staurosporine curve was used as positive control. We measured cytotoxicity with a fluorescence plate format assay that uses a cyanine dye impermeant to live cells that stains DNA from dead cells and increases its fluorescence. To further analyze the mechanism of cell death, we measured the release of lactate dehydrogenase LDH from cells with damaged membranes into the medium with the CytoTox-ONE homogeneous membrane integrity assay.
This is typically associated with cell necrosis Fig. We also measured release from cells of the necrosis marker high mobility group box 1 protein HMGB1 by western blot [ 26 ]. After attachment cells were treated with increasing concentrations of a AGK2 and b AK1 and incubated for 24 hours. Cytotoxicity was measure in a plate reader. Although SIRT2 has been considered a potential regulator of cell cycle progression [ 28 – 30 ], we did not find evidence for that mechanism of action in this study.
SIRT2 was reported to associate with mitotic structures and to increase in abundance and phosphorylation during mitosis.
It has been reported to prevent chromosome condensation and entry into M phase in response to mitotic stress. In Schwann cells, selective inactivation of Sirt2 during early embryogenesis did not reduce the number of Schwann cells produced during the period of rapid cell proliferation that occurs at post-natal days This supports the conclusion that cell cycle progression in Schwann cells is independent of SIRT2 activity [ 9 ] in agreement with our finding that SIRT2 inhibition did not decrease merlin-mutant MSC viability by arresting cell cycle progression but rather by inducing cell necrosis.
There is evidence for a role of SIRT2 in necrosis.
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Similar to our findings in Schwannoma and control Schwann cells, upregulation of SIRT2 mRNA and protein levels has luscheer reported in some cancer cells such as primary acute df leukemia blasts compared to control hematopoietic progenitor cells from healthy individuals [ 36 ]. Additionally, aged mice with inactivation of the Sirt2 gene by deletion of exons developed different types of tumors depending on their gender [ 19 ]. Similarly, another SIRT2 inhibitor, compound AC showed selectively decreasing viability in several human cancer cell lines over their human primary cells controls [ 21 ].
The panel included cell lines from prostate, pancreas, cervical and lung cancer.
AC was also selectively cytotoxic to HeLa cells by inducing apoptosis and necrosis. Discrepancies in the positive and negative associations of SIRT2 with tumor development are likely due to differences in cell type, developmental activation patterns for SIRT2 and substrate preferences [ 937 ].
For instance histone H4 is a principal substrate for SIRT2 in various cell types but not in oligodendrocytes [ 11 luscehr, 38 ]. Therefore it is possible that at the IC luschr concentrations used in this study, the compounds could have also slightly decreased SIRT1 and 3 activity. Both in vitro and in vivo treatment showed a selective anti-proliferative effect on schwannoma and meningioma cells warranting further clinical evaluation for NF2 related tumors [ 42 – 44 ].
We speculate that SIRT2 pharmacological inhibition may have some therapeutic value for NF2-associated schwannomas by promoting necrosis. However, additional research is needed to understand the relationship between merlin and SIRT2 in normal Schwann cells and the effect of merlin inactivation on protein dedcargar and cell survival. Fluorescence signals were acquired on separate channels with identical parameters for each labeled protein from a single plane. Fluorescence intensity of the green channel was done using Volocity software.
Pictures were processed with the ZEN software in the same manner. Viability of dose-response assays was assessed with the CellTiter-Fluor cell viability assay Promega following manufacturer’s specifications. Plates were quick spun again and returned to the incubator for 24 hours.
For the 72 hours cell proliferation study, cell numbers were assessed with the Crystal Violet Assay as previously described [ 46 ]. Equal number of merlin-mutant MSC were seeded in well plates and cell number was evaluated at 0; 24; 48 and 72 hour time points.
Both assay were previously described [ 23 ]. No stop solution was used. When media was removed from cultures, the plates were washed once with ice cold PBS and cell were lysed with the modified RIPA buffer mentioned above; protein concentration was measured with the DC assay BioRad. GraphPad Prism version 5.
AGK-2 and AK1 dose-response experiments were analyzed by non-linear regression four parameters. Experimental data from three independent experiments were statistically analyzed by one or two-way ANOVA with post-tests, as indicated for each experiment. The authors reported no potential conflicts of interest.
National Center for Biotechnology InformationU. Journal List Oncotarget v. Published online Nov Author information Article notes Copyright and License information Disclaimer. Received Sep 18; Accepted Nov This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. Associated Data Supplementary Materials oncotargets Abstract Mutations in the NF2 gene cause Neurofibromatosis Type 2 NF2a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Open in a separate window. SIRT2 in Cancer Similar to our findings in Schwannoma and control Schwann cells, upregulation of SIRT2 mRNA and protein levels has been reported in some cancer cells such as primary acute myeloid leukemia blasts compared to control hematopoietic progenitor cells from healthy individuals [ 36 ].
Cell Viability Assay Viability of dose-response assays was assessed with the CellTiter-Fluor cell viability assay Promega following manufacturer’s specifications. Proliferation Assays For the 72 hours cell proliferation study, cell numbers were assessed with the Crystal Violet Assay as previously described [ 46 ].
Statistical Analysis GraphPad Prism version 5. Supplemental Figure Click here to view.