Limits...
LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

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Inhibition or silencing of LIMK does not induce apoptosis.(a) Caspase 3/7 activity assay. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth media, phenol-red free in a 384-well plate and incubated for 16 hrs at 37°C, 7% CO2. Next, 5 µl/well of the indicated solution was added and incubated for 8 hrs. Caspase 3/7 activity was measured with the ApoONE Homogeneous assay. Staurosporine curve was used as positive control. Histogram represents 3 independent experiments (n=96) normalized to untreated and analyzed together. **P<0.01; ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (b)Nf2ΔEx2 MSCs plated in a 6-well format were incubated with 2 µM BMS-5 or DMSO vehicle for 24 hrs. Plasma membrane asymmetry was evaluated with the Violet ratiometric assay by flow cytometry. Densitometry graph illustrates the following quadrants: apoptotic (Q3), dead (Q1), live (Q4) and dual label (Q2). (c) Caspase 3/7 activity measured with the ApoONE assay of Nf2ΔEx2 MSCs uninfected or infected with control scrambled or the indicated constructs for LIMK1/2 shRNA silencing. Cells were incubated 24 hrs in a 384-well format and in the presence of inhibitors for 8 hrs where indicated. Staurosporine response was used as positive control. Histogram represents 5 independent experiments normalized to scrambled control and analyzed together (n=160). ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (d)Nf2ΔEx2 MSCs infected with scrambled shRNA or shRNAs targeting LIMK1/2 constructs were plated in a 6-well format and incubated for 24 hrs. Plasma membrane asymmetry was assessed with the Violet ratiometric assay by flow cytometry in three independent experiments. Densitometry plots show the quantification of the population distribution into the four quadrants as in (b).
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Figure 4: Inhibition or silencing of LIMK does not induce apoptosis.(a) Caspase 3/7 activity assay. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth media, phenol-red free in a 384-well plate and incubated for 16 hrs at 37°C, 7% CO2. Next, 5 µl/well of the indicated solution was added and incubated for 8 hrs. Caspase 3/7 activity was measured with the ApoONE Homogeneous assay. Staurosporine curve was used as positive control. Histogram represents 3 independent experiments (n=96) normalized to untreated and analyzed together. **P<0.01; ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (b)Nf2ΔEx2 MSCs plated in a 6-well format were incubated with 2 µM BMS-5 or DMSO vehicle for 24 hrs. Plasma membrane asymmetry was evaluated with the Violet ratiometric assay by flow cytometry. Densitometry graph illustrates the following quadrants: apoptotic (Q3), dead (Q1), live (Q4) and dual label (Q2). (c) Caspase 3/7 activity measured with the ApoONE assay of Nf2ΔEx2 MSCs uninfected or infected with control scrambled or the indicated constructs for LIMK1/2 shRNA silencing. Cells were incubated 24 hrs in a 384-well format and in the presence of inhibitors for 8 hrs where indicated. Staurosporine response was used as positive control. Histogram represents 5 independent experiments normalized to scrambled control and analyzed together (n=160). ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (d)Nf2ΔEx2 MSCs infected with scrambled shRNA or shRNAs targeting LIMK1/2 constructs were plated in a 6-well format and incubated for 24 hrs. Plasma membrane asymmetry was assessed with the Violet ratiometric assay by flow cytometry in three independent experiments. Densitometry plots show the quantification of the population distribution into the four quadrants as in (b).

Mentions: To determine if BMS-5 promoted Nf2ΔEx2 MSCs apoptosis, we measured caspase 3/7 activity. We found that BMS-5 did not activate caspase3/7, whereas the positive control, staurosporine, promoted dose-dependent activation of caspase 3/7 (Figure 5 a). To determine if BMS-5 promoted caspase-independent apoptosis, we analyzed BMS-5-treated Nf2ΔEx2 MSCs by flow cytometry using a ratiometric membrane asymmetry probe that detects loss of membrane phospholipid asymmetry associated with apoptosis. We observed only a small increase (3.5%) of apoptotic cells after 7 hours of BMS-5 treatment (Figure 4 b). Consistent with pharmacological LIMK inhibition, knockdown of LIMK1 or LIMK2 was not associated with caspase 3/7 activity increase compared to scrambled shRNA controls examined 24 hours after plating (Figure 4 c). Similarly, caspase-independent apoptosis was not observed in Nf2ΔEx2 MSCs transduced with either LIMK1 or LIMK2 shRNA (Figure 4 d). These results suggest that pharmacological inhibition and genetic silencing of LIMK affect Nf2ΔEx2 MSC viability through an apoptosis-independent mechanism.


LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Inhibition or silencing of LIMK does not induce apoptosis.(a) Caspase 3/7 activity assay. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth media, phenol-red free in a 384-well plate and incubated for 16 hrs at 37°C, 7% CO2. Next, 5 µl/well of the indicated solution was added and incubated for 8 hrs. Caspase 3/7 activity was measured with the ApoONE Homogeneous assay. Staurosporine curve was used as positive control. Histogram represents 3 independent experiments (n=96) normalized to untreated and analyzed together. **P<0.01; ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (b)Nf2ΔEx2 MSCs plated in a 6-well format were incubated with 2 µM BMS-5 or DMSO vehicle for 24 hrs. Plasma membrane asymmetry was evaluated with the Violet ratiometric assay by flow cytometry. Densitometry graph illustrates the following quadrants: apoptotic (Q3), dead (Q1), live (Q4) and dual label (Q2). (c) Caspase 3/7 activity measured with the ApoONE assay of Nf2ΔEx2 MSCs uninfected or infected with control scrambled or the indicated constructs for LIMK1/2 shRNA silencing. Cells were incubated 24 hrs in a 384-well format and in the presence of inhibitors for 8 hrs where indicated. Staurosporine response was used as positive control. Histogram represents 5 independent experiments normalized to scrambled control and analyzed together (n=160). ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (d)Nf2ΔEx2 MSCs infected with scrambled shRNA or shRNAs targeting LIMK1/2 constructs were plated in a 6-well format and incubated for 24 hrs. Plasma membrane asymmetry was assessed with the Violet ratiometric assay by flow cytometry in three independent experiments. Densitometry plots show the quantification of the population distribution into the four quadrants as in (b).
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Figure 4: Inhibition or silencing of LIMK does not induce apoptosis.(a) Caspase 3/7 activity assay. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth media, phenol-red free in a 384-well plate and incubated for 16 hrs at 37°C, 7% CO2. Next, 5 µl/well of the indicated solution was added and incubated for 8 hrs. Caspase 3/7 activity was measured with the ApoONE Homogeneous assay. Staurosporine curve was used as positive control. Histogram represents 3 independent experiments (n=96) normalized to untreated and analyzed together. **P<0.01; ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (b)Nf2ΔEx2 MSCs plated in a 6-well format were incubated with 2 µM BMS-5 or DMSO vehicle for 24 hrs. Plasma membrane asymmetry was evaluated with the Violet ratiometric assay by flow cytometry. Densitometry graph illustrates the following quadrants: apoptotic (Q3), dead (Q1), live (Q4) and dual label (Q2). (c) Caspase 3/7 activity measured with the ApoONE assay of Nf2ΔEx2 MSCs uninfected or infected with control scrambled or the indicated constructs for LIMK1/2 shRNA silencing. Cells were incubated 24 hrs in a 384-well format and in the presence of inhibitors for 8 hrs where indicated. Staurosporine response was used as positive control. Histogram represents 5 independent experiments normalized to scrambled control and analyzed together (n=160). ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test. (d)Nf2ΔEx2 MSCs infected with scrambled shRNA or shRNAs targeting LIMK1/2 constructs were plated in a 6-well format and incubated for 24 hrs. Plasma membrane asymmetry was assessed with the Violet ratiometric assay by flow cytometry in three independent experiments. Densitometry plots show the quantification of the population distribution into the four quadrants as in (b).
Mentions: To determine if BMS-5 promoted Nf2ΔEx2 MSCs apoptosis, we measured caspase 3/7 activity. We found that BMS-5 did not activate caspase3/7, whereas the positive control, staurosporine, promoted dose-dependent activation of caspase 3/7 (Figure 5 a). To determine if BMS-5 promoted caspase-independent apoptosis, we analyzed BMS-5-treated Nf2ΔEx2 MSCs by flow cytometry using a ratiometric membrane asymmetry probe that detects loss of membrane phospholipid asymmetry associated with apoptosis. We observed only a small increase (3.5%) of apoptotic cells after 7 hours of BMS-5 treatment (Figure 4 b). Consistent with pharmacological LIMK inhibition, knockdown of LIMK1 or LIMK2 was not associated with caspase 3/7 activity increase compared to scrambled shRNA controls examined 24 hours after plating (Figure 4 c). Similarly, caspase-independent apoptosis was not observed in Nf2ΔEx2 MSCs transduced with either LIMK1 or LIMK2 shRNA (Figure 4 d). These results suggest that pharmacological inhibition and genetic silencing of LIMK affect Nf2ΔEx2 MSC viability through an apoptosis-independent mechanism.

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Show MeSH
Related in: MedlinePlus