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Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence.

Liu Y, Hawkins OE, Su Y, Vilgelm AE, Sobolik T, Thu YM, Kantrow S, Splittgerber RC, Short S, Amiri KI, Ecsedy JA, Sosman JA, Kelley MC, Richmond A - EMBO Mol Med (2012)

Bottom Line: Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP).Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP.Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA.

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Disruption of IKKβ/NF-κB compromises drug-induced senescenceHs294T cells were transfected with IKKβ siRNA, and knockdown of IKKβ verified by Western blot. siRNA control or siIKKβ transfected cells were treated with 1 µM MLN8237 for 5 days, and senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with the IKKβ inhibitor BMS-345541 (10 µM) for 48 h, and the level of p-p65 and p65 were analysed by Western blot.Hs294T cells were treated with 1 µM MLN8237 with or without 10 µM BMS-34554 for 5 days. After treatment, viable cells were counted and 5 × 105 cells were seeded into 10-cm plates in DMEM F-12 with 10% FBS. Once cells attached, serum-containing medium was replaced with serum-free medium and cells were cultured overnight. Cytokine secretion into the medium was assayed by cytokine array.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 2 days and DNA content was examined by FACS.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days. After treatment, senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days, and the viable cells were counted using a haemocytometer. Data indicate mean values ± SD (n = 3). With the exception of Fig 8C, all experiments were performed in triplicate with high reproducibility and representative experiments are shown.
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fig08: Disruption of IKKβ/NF-κB compromises drug-induced senescenceHs294T cells were transfected with IKKβ siRNA, and knockdown of IKKβ verified by Western blot. siRNA control or siIKKβ transfected cells were treated with 1 µM MLN8237 for 5 days, and senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with the IKKβ inhibitor BMS-345541 (10 µM) for 48 h, and the level of p-p65 and p65 were analysed by Western blot.Hs294T cells were treated with 1 µM MLN8237 with or without 10 µM BMS-34554 for 5 days. After treatment, viable cells were counted and 5 × 105 cells were seeded into 10-cm plates in DMEM F-12 with 10% FBS. Once cells attached, serum-containing medium was replaced with serum-free medium and cells were cultured overnight. Cytokine secretion into the medium was assayed by cytokine array.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 2 days and DNA content was examined by FACS.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days. After treatment, senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days, and the viable cells were counted using a haemocytometer. Data indicate mean values ± SD (n = 3). With the exception of Fig 8C, all experiments were performed in triplicate with high reproducibility and representative experiments are shown.

Mentions: To address the significance of NF-κB activation on treatment outcome, we knocked down IKKβ to reduce NF-κB activity and observed that aurora kinase inhibitor-induced senescence was impaired (Fig 8A). IKKβ stable-knockdown cells gave rise to a similar phenotype (Supporting Information Fig S14). We also confirmed our results using the IKKβ inhibitor BMS-345541 (10 µM) to block the NF-κB p65 pathway (Fig 8B). When IKKβ activity was suppressed, the MLN8237-induced SASP was decreased (Fig 8C), polyploidy was reduced (Fig 8D), and less senescence was observed (Fig 8E). Although targeting IKKβ/NF-κB with BMS-345541 induces apoptosis in melanoma cells (Yang et al, 2006b), we did not observe synergistic effects on cell growth/survival when BMS345541 was combined with MLN8237 in vitro (Fig 8F), likely because blocking IKKβ reduces the induction of senescence by MLN8237, so the effect of combined treatment is largely the result of apoptosis induction by inhibition of IKKβ.


Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence.

Liu Y, Hawkins OE, Su Y, Vilgelm AE, Sobolik T, Thu YM, Kantrow S, Splittgerber RC, Short S, Amiri KI, Ecsedy JA, Sosman JA, Kelley MC, Richmond A - EMBO Mol Med (2012)

Disruption of IKKβ/NF-κB compromises drug-induced senescenceHs294T cells were transfected with IKKβ siRNA, and knockdown of IKKβ verified by Western blot. siRNA control or siIKKβ transfected cells were treated with 1 µM MLN8237 for 5 days, and senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with the IKKβ inhibitor BMS-345541 (10 µM) for 48 h, and the level of p-p65 and p65 were analysed by Western blot.Hs294T cells were treated with 1 µM MLN8237 with or without 10 µM BMS-34554 for 5 days. After treatment, viable cells were counted and 5 × 105 cells were seeded into 10-cm plates in DMEM F-12 with 10% FBS. Once cells attached, serum-containing medium was replaced with serum-free medium and cells were cultured overnight. Cytokine secretion into the medium was assayed by cytokine array.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 2 days and DNA content was examined by FACS.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days. After treatment, senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days, and the viable cells were counted using a haemocytometer. Data indicate mean values ± SD (n = 3). With the exception of Fig 8C, all experiments were performed in triplicate with high reproducibility and representative experiments are shown.
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Related In: Results  -  Collection

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fig08: Disruption of IKKβ/NF-κB compromises drug-induced senescenceHs294T cells were transfected with IKKβ siRNA, and knockdown of IKKβ verified by Western blot. siRNA control or siIKKβ transfected cells were treated with 1 µM MLN8237 for 5 days, and senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with the IKKβ inhibitor BMS-345541 (10 µM) for 48 h, and the level of p-p65 and p65 were analysed by Western blot.Hs294T cells were treated with 1 µM MLN8237 with or without 10 µM BMS-34554 for 5 days. After treatment, viable cells were counted and 5 × 105 cells were seeded into 10-cm plates in DMEM F-12 with 10% FBS. Once cells attached, serum-containing medium was replaced with serum-free medium and cells were cultured overnight. Cytokine secretion into the medium was assayed by cytokine array.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 2 days and DNA content was examined by FACS.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days. After treatment, senescence was evaluated by β-galactosidase staining.Hs294T and SK-Mel-28 cells were treated with 10 µM BMS-345541, 1 µM MLN8237, or both for 5 days, and the viable cells were counted using a haemocytometer. Data indicate mean values ± SD (n = 3). With the exception of Fig 8C, all experiments were performed in triplicate with high reproducibility and representative experiments are shown.
Mentions: To address the significance of NF-κB activation on treatment outcome, we knocked down IKKβ to reduce NF-κB activity and observed that aurora kinase inhibitor-induced senescence was impaired (Fig 8A). IKKβ stable-knockdown cells gave rise to a similar phenotype (Supporting Information Fig S14). We also confirmed our results using the IKKβ inhibitor BMS-345541 (10 µM) to block the NF-κB p65 pathway (Fig 8B). When IKKβ activity was suppressed, the MLN8237-induced SASP was decreased (Fig 8C), polyploidy was reduced (Fig 8D), and less senescence was observed (Fig 8E). Although targeting IKKβ/NF-κB with BMS-345541 induces apoptosis in melanoma cells (Yang et al, 2006b), we did not observe synergistic effects on cell growth/survival when BMS345541 was combined with MLN8237 in vitro (Fig 8F), likely because blocking IKKβ reduces the induction of senescence by MLN8237, so the effect of combined treatment is largely the result of apoptosis induction by inhibition of IKKβ.

Bottom Line: Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP).Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP.Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA.

Show MeSH
Related in: MedlinePlus