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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LIMK inhibition by BMS-5 or silencing by shRNA in Nf2ΔEx2 MSCs decreases cell viability. (a) BMS-5 dose-response western blot. Nf2ΔEx2 MSCs were plated in 12-well plates. Cultures were treated the next day as indicated for 30 min. Cells were harvested, lysed and analyzed by western blotting for phospho-Ser3-cofilin and total cofilin. β-actin levels were used as loading controls for normalization. Representative blot of three independent experiments. (b) BMS-5 dose-response curve of cofilin phosphorylation in Nf2ΔEx2 MSCs. Analyzed as log [inhibitor] vs. response, variable slope (four parameters). (c) BMS-5 dose viability-response curve. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth medium phenol-red free in a 384-well plate and after attachment were incubated with BMS-5 for 24 hrs. Cell viability was measured with the CellTiter-Fluor assay. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96) log [inhibitor] vs. response, variable slope (four parameters). (d) BMS-5 viability response of control MSCs. Cell viability was measured as in (c). Graph represents the mean ± SEM (n=16). DMSO control was considered 100% viability. Rapamycin (RM) (50 µM) was a positive control for cell death. ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.(e) Viability of Nf2ΔEx2 MSCs expressing LIMK 1 or LIMK2 shRNAs were compared to cells expressing scrambled shRNA untreated or treated with 5 µM BMS-5 or 50 µM rapamycin (RM) for 24 hrs in a 384-well format. Cell viability was measured with the CellTiter-Fluor assay. Nf2ΔEx2 MSCs expressing scrambled shRNA represented 100% viability, and the 50 µM RM treated cells were a positive control for cell death. Graph represents the mean ± SEM of 4 independent experiments analyzed together (n=128). ***P<0.001determined by one-way ANOVA using Dunnett’s multiple comparison test
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Figure 3: LIMK inhibition by BMS-5 or silencing by shRNA in Nf2ΔEx2 MSCs decreases cell viability. (a) BMS-5 dose-response western blot. Nf2ΔEx2 MSCs were plated in 12-well plates. Cultures were treated the next day as indicated for 30 min. Cells were harvested, lysed and analyzed by western blotting for phospho-Ser3-cofilin and total cofilin. β-actin levels were used as loading controls for normalization. Representative blot of three independent experiments. (b) BMS-5 dose-response curve of cofilin phosphorylation in Nf2ΔEx2 MSCs. Analyzed as log [inhibitor] vs. response, variable slope (four parameters). (c) BMS-5 dose viability-response curve. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth medium phenol-red free in a 384-well plate and after attachment were incubated with BMS-5 for 24 hrs. Cell viability was measured with the CellTiter-Fluor assay. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96) log [inhibitor] vs. response, variable slope (four parameters). (d) BMS-5 viability response of control MSCs. Cell viability was measured as in (c). Graph represents the mean ± SEM (n=16). DMSO control was considered 100% viability. Rapamycin (RM) (50 µM) was a positive control for cell death. ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.(e) Viability of Nf2ΔEx2 MSCs expressing LIMK 1 or LIMK2 shRNAs were compared to cells expressing scrambled shRNA untreated or treated with 5 µM BMS-5 or 50 µM rapamycin (RM) for 24 hrs in a 384-well format. Cell viability was measured with the CellTiter-Fluor assay. Nf2ΔEx2 MSCs expressing scrambled shRNA represented 100% viability, and the 50 µM RM treated cells were a positive control for cell death. Graph represents the mean ± SEM of 4 independent experiments analyzed together (n=128). ***P<0.001determined by one-way ANOVA using Dunnett’s multiple comparison test

Mentions: We next sought to determine whether LIMK was a potential drug target for NF2. We tested the efficacy of BMS-5, a highly selective small molecule inhibitor of LIMK1/2, on reducing phosphorylation of cofilin on Ser3 (ref. 35). BMS-5 inhibited cofilin-Ser3 phosphorylation in a dose-dependent manner in Nf2ΔEx2 MSCs with an IC50 of ~ 2 µM (Figure 3 a, b). We next tested the ability of BMS-5 to reduce viability of Nf2ΔEx2 MSCs. The 100% viability control was 0.1% DMSO and a positive cell death control was obtained with 50 µM rapamycin (RM) that caused approximately 80% cell death in 24 hours. BMS-5 reduced Nf2ΔEx2 MSC viability in a dose-dependent manner with an IC50 of 3.9 µM (Figure 3 c), but did not significantly reduce the viability of control Nf2flox2/flox2 MSCs at equivalent BMS-5 concentrations (Figure 3 d). At 10µM BMS-5, Nf2∆Ex2 MSC viability was 40% compared to 83% for controls.


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)

LIMK inhibition by BMS-5 or silencing by shRNA in Nf2ΔEx2 MSCs decreases cell viability. (a) BMS-5 dose-response western blot. Nf2ΔEx2 MSCs were plated in 12-well plates. Cultures were treated the next day as indicated for 30 min. Cells were harvested, lysed and analyzed by western blotting for phospho-Ser3-cofilin and total cofilin. β-actin levels were used as loading controls for normalization. Representative blot of three independent experiments. (b) BMS-5 dose-response curve of cofilin phosphorylation in Nf2ΔEx2 MSCs. Analyzed as log [inhibitor] vs. response, variable slope (four parameters). (c) BMS-5 dose viability-response curve. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth medium phenol-red free in a 384-well plate and after attachment were incubated with BMS-5 for 24 hrs. Cell viability was measured with the CellTiter-Fluor assay. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96) log [inhibitor] vs. response, variable slope (four parameters). (d) BMS-5 viability response of control MSCs. Cell viability was measured as in (c). Graph represents the mean ± SEM (n=16). DMSO control was considered 100% viability. Rapamycin (RM) (50 µM) was a positive control for cell death. ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.(e) Viability of Nf2ΔEx2 MSCs expressing LIMK 1 or LIMK2 shRNAs were compared to cells expressing scrambled shRNA untreated or treated with 5 µM BMS-5 or 50 µM rapamycin (RM) for 24 hrs in a 384-well format. Cell viability was measured with the CellTiter-Fluor assay. Nf2ΔEx2 MSCs expressing scrambled shRNA represented 100% viability, and the 50 µM RM treated cells were a positive control for cell death. Graph represents the mean ± SEM of 4 independent experiments analyzed together (n=128). ***P<0.001determined by one-way ANOVA using Dunnett’s multiple comparison test
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016185&req=5

Figure 3: LIMK inhibition by BMS-5 or silencing by shRNA in Nf2ΔEx2 MSCs decreases cell viability. (a) BMS-5 dose-response western blot. Nf2ΔEx2 MSCs were plated in 12-well plates. Cultures were treated the next day as indicated for 30 min. Cells were harvested, lysed and analyzed by western blotting for phospho-Ser3-cofilin and total cofilin. β-actin levels were used as loading controls for normalization. Representative blot of three independent experiments. (b) BMS-5 dose-response curve of cofilin phosphorylation in Nf2ΔEx2 MSCs. Analyzed as log [inhibitor] vs. response, variable slope (four parameters). (c) BMS-5 dose viability-response curve. Nf2ΔEx2 MSCs were seeded at 5 000 cells/well in 20 µl growth medium phenol-red free in a 384-well plate and after attachment were incubated with BMS-5 for 24 hrs. Cell viability was measured with the CellTiter-Fluor assay. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96) log [inhibitor] vs. response, variable slope (four parameters). (d) BMS-5 viability response of control MSCs. Cell viability was measured as in (c). Graph represents the mean ± SEM (n=16). DMSO control was considered 100% viability. Rapamycin (RM) (50 µM) was a positive control for cell death. ***P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.(e) Viability of Nf2ΔEx2 MSCs expressing LIMK 1 or LIMK2 shRNAs were compared to cells expressing scrambled shRNA untreated or treated with 5 µM BMS-5 or 50 µM rapamycin (RM) for 24 hrs in a 384-well format. Cell viability was measured with the CellTiter-Fluor assay. Nf2ΔEx2 MSCs expressing scrambled shRNA represented 100% viability, and the 50 µM RM treated cells were a positive control for cell death. Graph represents the mean ± SEM of 4 independent experiments analyzed together (n=128). ***P<0.001determined by one-way ANOVA using Dunnett’s multiple comparison test
Mentions: We next sought to determine whether LIMK was a potential drug target for NF2. We tested the efficacy of BMS-5, a highly selective small molecule inhibitor of LIMK1/2, on reducing phosphorylation of cofilin on Ser3 (ref. 35). BMS-5 inhibited cofilin-Ser3 phosphorylation in a dose-dependent manner in Nf2ΔEx2 MSCs with an IC50 of ~ 2 µM (Figure 3 a, b). We next tested the ability of BMS-5 to reduce viability of Nf2ΔEx2 MSCs. The 100% viability control was 0.1% DMSO and a positive cell death control was obtained with 50 µM rapamycin (RM) that caused approximately 80% cell death in 24 hours. BMS-5 reduced Nf2ΔEx2 MSC viability in a dose-dependent manner with an IC50 of 3.9 µM (Figure 3 c), but did not significantly reduce the viability of control Nf2flox2/flox2 MSCs at equivalent BMS-5 concentrations (Figure 3 d). At 10µM BMS-5, Nf2∆Ex2 MSC viability was 40% compared to 83% for controls.

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