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Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

Petrilli A, Bott M, Fernández-Valle C - Oncotarget (2013)

Bottom Line: Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability.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.These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Lake Nona-Orlando, Florida, USA.

ABSTRACT
Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling 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. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. 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. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

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Merlin-Mutant MSC (M-mut MSC) Have Lower Levels of Lysine Acetylation and Higher Levels of SIRT2 Compared to Control MSCControl Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for: a) Acetyl-lysine, b) SIRT2, acetyl-α-tubulin, α-tubulin and c) SIRT2, GAPDH. Anti-β-actin was used as a loading control. d) Cultured control human Schwann cells (HSCs) from normal individuals and HEI193 cells were analyzed by western blotting for SIRT2, acetyl-α-tubulin, α-tubulin. Anti-β-actin was used as a loading control. e) Representative confocal images of control MSC and merlin-mutant MSC grown overnight on glass coverslips and immunostained with the indicated antibodies (green). The nucleus was visualized with DAPI stain (blue) and F-actin with phalloidin-Alexa633 (white). Scale bar: 20 μm. Quantitation of the immunofluorescence from three independent experiments was performed with Volocity software. ****P<0.0001 determined by two-way ANOVA using Bonferroni post-tests. f) Control Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for SIRT1, SIRT2, SIRT3, SIRT5, SIRT7 and β-actin.
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Figure 1: Merlin-Mutant MSC (M-mut MSC) Have Lower Levels of Lysine Acetylation and Higher Levels of SIRT2 Compared to Control MSCControl Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for: a) Acetyl-lysine, b) SIRT2, acetyl-α-tubulin, α-tubulin and c) SIRT2, GAPDH. Anti-β-actin was used as a loading control. d) Cultured control human Schwann cells (HSCs) from normal individuals and HEI193 cells were analyzed by western blotting for SIRT2, acetyl-α-tubulin, α-tubulin. Anti-β-actin was used as a loading control. e) Representative confocal images of control MSC and merlin-mutant MSC grown overnight on glass coverslips and immunostained with the indicated antibodies (green). The nucleus was visualized with DAPI stain (blue) and F-actin with phalloidin-Alexa633 (white). Scale bar: 20 μm. Quantitation of the immunofluorescence from three independent experiments was performed with Volocity software. ****P<0.0001 determined by two-way ANOVA using Bonferroni post-tests. f) Control Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for SIRT1, SIRT2, SIRT3, SIRT5, SIRT7 and β-actin.

Mentions: To visualize the lysine acetylation patterns in merlin-mutant MSC and control MSC, we performed a western blot using an ε-acetyl-Lysine antibody. Control MSC had a higher number of and more intensely acetylated bands than merlin-mutant MSC (Fig. 1a and Supplementary Fig. S1). Because the merlin-mutant MSC presented fewer bands, we asked whether SIRT2, the target of AGK2 had altered expression levels. Indeed we found that merlin-mutant MSC expressed SIRT2 at higher levels than control MSC (Fig. 1b). The acetyl-lysine blot differed greatly around the 50 kDa molecular weight marker. Because SIRT2 is a recognized acetyl-α-tubulin deacetylase, we investigated if any of those bands were α-tubulin. Merlin-mutant MSC have highly deacetylated tubulin levels compared to control MSC (Fig. 1b). We confirmed these differences with several independently derived merlin-mutant MSC lines (Fig. 1c). We next immunoblotted for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) because tumor cells have altered metabolism and sirtuins can influence metabolic and energetic regulation. We found higher levels of GAPDH in merlin-mutant MSC compared to MSC consistent with higher glycolytic activity in merlin-mutant MSC than controls (Fig. 1c). In order to validate the results in our cellular model, we measured SIRT2 and acetyl-α-tubulin levels in a human NF2 cell line created by immortalization of schwannoma cells from a NF2 patient with the E6 and E7 genes of the papillomavirus [25]. Similarly, we found higher levels of SIRT2 in HEI-193 cells than in control human Schwann cells which correlated with lower acetylated tubulin levels in HEI-193 compared to control cells (Fig. 1d).


Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

Petrilli A, Bott M, Fernández-Valle C - Oncotarget (2013)

Merlin-Mutant MSC (M-mut MSC) Have Lower Levels of Lysine Acetylation and Higher Levels of SIRT2 Compared to Control MSCControl Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for: a) Acetyl-lysine, b) SIRT2, acetyl-α-tubulin, α-tubulin and c) SIRT2, GAPDH. Anti-β-actin was used as a loading control. d) Cultured control human Schwann cells (HSCs) from normal individuals and HEI193 cells were analyzed by western blotting for SIRT2, acetyl-α-tubulin, α-tubulin. Anti-β-actin was used as a loading control. e) Representative confocal images of control MSC and merlin-mutant MSC grown overnight on glass coverslips and immunostained with the indicated antibodies (green). The nucleus was visualized with DAPI stain (blue) and F-actin with phalloidin-Alexa633 (white). Scale bar: 20 μm. Quantitation of the immunofluorescence from three independent experiments was performed with Volocity software. ****P<0.0001 determined by two-way ANOVA using Bonferroni post-tests. f) Control Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for SIRT1, SIRT2, SIRT3, SIRT5, SIRT7 and β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926832&req=5

Figure 1: Merlin-Mutant MSC (M-mut MSC) Have Lower Levels of Lysine Acetylation and Higher Levels of SIRT2 Compared to Control MSCControl Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for: a) Acetyl-lysine, b) SIRT2, acetyl-α-tubulin, α-tubulin and c) SIRT2, GAPDH. Anti-β-actin was used as a loading control. d) Cultured control human Schwann cells (HSCs) from normal individuals and HEI193 cells were analyzed by western blotting for SIRT2, acetyl-α-tubulin, α-tubulin. Anti-β-actin was used as a loading control. e) Representative confocal images of control MSC and merlin-mutant MSC grown overnight on glass coverslips and immunostained with the indicated antibodies (green). The nucleus was visualized with DAPI stain (blue) and F-actin with phalloidin-Alexa633 (white). Scale bar: 20 μm. Quantitation of the immunofluorescence from three independent experiments was performed with Volocity software. ****P<0.0001 determined by two-way ANOVA using Bonferroni post-tests. f) Control Nf2flox2/flox2 MSC and merlin-mutant MSC were analyzed by western blotting for SIRT1, SIRT2, SIRT3, SIRT5, SIRT7 and β-actin.
Mentions: To visualize the lysine acetylation patterns in merlin-mutant MSC and control MSC, we performed a western blot using an ε-acetyl-Lysine antibody. Control MSC had a higher number of and more intensely acetylated bands than merlin-mutant MSC (Fig. 1a and Supplementary Fig. S1). Because the merlin-mutant MSC presented fewer bands, we asked whether SIRT2, the target of AGK2 had altered expression levels. Indeed we found that merlin-mutant MSC expressed SIRT2 at higher levels than control MSC (Fig. 1b). The acetyl-lysine blot differed greatly around the 50 kDa molecular weight marker. Because SIRT2 is a recognized acetyl-α-tubulin deacetylase, we investigated if any of those bands were α-tubulin. Merlin-mutant MSC have highly deacetylated tubulin levels compared to control MSC (Fig. 1b). We confirmed these differences with several independently derived merlin-mutant MSC lines (Fig. 1c). We next immunoblotted for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) because tumor cells have altered metabolism and sirtuins can influence metabolic and energetic regulation. We found higher levels of GAPDH in merlin-mutant MSC compared to MSC consistent with higher glycolytic activity in merlin-mutant MSC than controls (Fig. 1c). In order to validate the results in our cellular model, we measured SIRT2 and acetyl-α-tubulin levels in a human NF2 cell line created by immortalization of schwannoma cells from a NF2 patient with the E6 and E7 genes of the papillomavirus [25]. Similarly, we found higher levels of SIRT2 in HEI-193 cells than in control human Schwann cells which correlated with lower acetylated tubulin levels in HEI-193 compared to control cells (Fig. 1d).

Bottom Line: Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability.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.These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Lake Nona-Orlando, Florida, USA.

ABSTRACT
Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling 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. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. 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. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

Show MeSH
Related in: MedlinePlus