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BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

Lee JJ, Lee JS, Cui MN, Yun HH, Kim HY, Lee SH, Lee JH - Cell Death Dis (2014)

Bottom Line: The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level.Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction.Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Korea [2] Catholic Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea.

ABSTRACT
Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression via an induction of senescence through the regulation of STAT3/SKP2/p27 pathway.

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14-3-3ζ is involved in BIS depletion-induced senescence through the regulation of the STAT3 pathway. (a) Western blot assay for 14-3-3ζ and 14-3-3θ following SiBIS (100 nM) treatment. (b) The expression levels of 14-3-3ζ, p-STAT3 (S727 and Y705), STAT3, SKP2 and p27 were examined at the indicated times following 14-3-3ζ knockdown. (c) 14-3-3ζ depletion-induced senescence as determined by SA-β-Gal activities. ***P<0.001 versus control cells. (d) The overexpression of 14-3-3ζ prevented BIS depletion-induced senescence. Bars represent mean±S.E. from triplicate experiments.The percentage of SA-β-Gal-positive cells is shown in the right column. ***P<0.001 versus SiBIS-only treated cells. Scale bars, 50 μm. (e) Immunoprecipitation and immunoblotting was performed with the indicated antibodies following the transfection of STAT3, 14-3-3ζ or BIS-expressing plasmid for 2 days
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fig5: 14-3-3ζ is involved in BIS depletion-induced senescence through the regulation of the STAT3 pathway. (a) Western blot assay for 14-3-3ζ and 14-3-3θ following SiBIS (100 nM) treatment. (b) The expression levels of 14-3-3ζ, p-STAT3 (S727 and Y705), STAT3, SKP2 and p27 were examined at the indicated times following 14-3-3ζ knockdown. (c) 14-3-3ζ depletion-induced senescence as determined by SA-β-Gal activities. ***P<0.001 versus control cells. (d) The overexpression of 14-3-3ζ prevented BIS depletion-induced senescence. Bars represent mean±S.E. from triplicate experiments.The percentage of SA-β-Gal-positive cells is shown in the right column. ***P<0.001 versus SiBIS-only treated cells. Scale bars, 50 μm. (e) Immunoprecipitation and immunoblotting was performed with the indicated antibodies following the transfection of STAT3, 14-3-3ζ or BIS-expressing plasmid for 2 days

Mentions: Recently, sequential systemic proteomic analyses demonstrated that BIS and STAT3 were enrolled in the lists of 14-3-3ζ interactome and, reversely, 14-3-3ζ was recognized as a BIS-binding protein in the BIS interactome.44, 45 The importance of 14-3-3ζ and BIS was described in promoting aggresome formation.46 Moreover, 14-3-3ζ was reported to interact with STAT3 in multiple myeloma cells.47 Based on these previous findings, we tested the possibility that 14-3-3ζ is involved in the coupling of BIS and STAT3 with respect to the regulation of senescence. Figure 5a shows a gradual decrease in 14-3-3ζ expression following the decrease in BIS, whereas the expression of 14-3-3θ, another 14-3-3 protein isoform, was not altered. We next evaluated if 14-3-3ζ silencing per se could trigger senescence. Immunoblotting analysis indicated that 14-3-3ζ-depleted cells exhibited similar profiles to BIS-depleted cells with regards to the expression of p-STAT3 (S727), total STAT3, SKP2 and p27, thus acting as a substitute for BIS depletion (Figure 5b). p-STAT3 (S727) levels but not p-STAT3 (Y705) were abolished upon 14-3-3ζ knockdown, which was in keeping with the previous report showing that 14-3-3ζ protects the dephosphorylation of S727 sites of STAT3 from PPA2 in multiple myeloma cells.47 In addition, the marked increase in the SA-β-Gal-positive cells was observed in 14-3-3ζ-depleted cells (Figure 5c). The SA-β-Gal assays also revealed that ectopic expression of 14-3-3ζ almost completely prevented senescence initiated by BIS depletion (Figure 5d). Taken together, these findings indicate that 14-3-3ζ expression is modulated by BIS and that 14-3-3ζ protein functions as a potent regulator of cellular senescence as well. Subsequent investigation into the physical association between BIS, 14-3-3ζ and STAT3 using immunoprecipitation and subsequent immunoblotting assays indicate that these proteins constitute one complex (Figure 5e).


BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

Lee JJ, Lee JS, Cui MN, Yun HH, Kim HY, Lee SH, Lee JH - Cell Death Dis (2014)

14-3-3ζ is involved in BIS depletion-induced senescence through the regulation of the STAT3 pathway. (a) Western blot assay for 14-3-3ζ and 14-3-3θ following SiBIS (100 nM) treatment. (b) The expression levels of 14-3-3ζ, p-STAT3 (S727 and Y705), STAT3, SKP2 and p27 were examined at the indicated times following 14-3-3ζ knockdown. (c) 14-3-3ζ depletion-induced senescence as determined by SA-β-Gal activities. ***P<0.001 versus control cells. (d) The overexpression of 14-3-3ζ prevented BIS depletion-induced senescence. Bars represent mean±S.E. from triplicate experiments.The percentage of SA-β-Gal-positive cells is shown in the right column. ***P<0.001 versus SiBIS-only treated cells. Scale bars, 50 μm. (e) Immunoprecipitation and immunoblotting was performed with the indicated antibodies following the transfection of STAT3, 14-3-3ζ or BIS-expressing plasmid for 2 days
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Related In: Results  -  Collection

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fig5: 14-3-3ζ is involved in BIS depletion-induced senescence through the regulation of the STAT3 pathway. (a) Western blot assay for 14-3-3ζ and 14-3-3θ following SiBIS (100 nM) treatment. (b) The expression levels of 14-3-3ζ, p-STAT3 (S727 and Y705), STAT3, SKP2 and p27 were examined at the indicated times following 14-3-3ζ knockdown. (c) 14-3-3ζ depletion-induced senescence as determined by SA-β-Gal activities. ***P<0.001 versus control cells. (d) The overexpression of 14-3-3ζ prevented BIS depletion-induced senescence. Bars represent mean±S.E. from triplicate experiments.The percentage of SA-β-Gal-positive cells is shown in the right column. ***P<0.001 versus SiBIS-only treated cells. Scale bars, 50 μm. (e) Immunoprecipitation and immunoblotting was performed with the indicated antibodies following the transfection of STAT3, 14-3-3ζ or BIS-expressing plasmid for 2 days
Mentions: Recently, sequential systemic proteomic analyses demonstrated that BIS and STAT3 were enrolled in the lists of 14-3-3ζ interactome and, reversely, 14-3-3ζ was recognized as a BIS-binding protein in the BIS interactome.44, 45 The importance of 14-3-3ζ and BIS was described in promoting aggresome formation.46 Moreover, 14-3-3ζ was reported to interact with STAT3 in multiple myeloma cells.47 Based on these previous findings, we tested the possibility that 14-3-3ζ is involved in the coupling of BIS and STAT3 with respect to the regulation of senescence. Figure 5a shows a gradual decrease in 14-3-3ζ expression following the decrease in BIS, whereas the expression of 14-3-3θ, another 14-3-3 protein isoform, was not altered. We next evaluated if 14-3-3ζ silencing per se could trigger senescence. Immunoblotting analysis indicated that 14-3-3ζ-depleted cells exhibited similar profiles to BIS-depleted cells with regards to the expression of p-STAT3 (S727), total STAT3, SKP2 and p27, thus acting as a substitute for BIS depletion (Figure 5b). p-STAT3 (S727) levels but not p-STAT3 (Y705) were abolished upon 14-3-3ζ knockdown, which was in keeping with the previous report showing that 14-3-3ζ protects the dephosphorylation of S727 sites of STAT3 from PPA2 in multiple myeloma cells.47 In addition, the marked increase in the SA-β-Gal-positive cells was observed in 14-3-3ζ-depleted cells (Figure 5c). The SA-β-Gal assays also revealed that ectopic expression of 14-3-3ζ almost completely prevented senescence initiated by BIS depletion (Figure 5d). Taken together, these findings indicate that 14-3-3ζ expression is modulated by BIS and that 14-3-3ζ protein functions as a potent regulator of cellular senescence as well. Subsequent investigation into the physical association between BIS, 14-3-3ζ and STAT3 using immunoprecipitation and subsequent immunoblotting assays indicate that these proteins constitute one complex (Figure 5e).

Bottom Line: The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level.Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction.Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Korea [2] Catholic Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea.

ABSTRACT
Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression via an induction of senescence through the regulation of STAT3/SKP2/p27 pathway.

Show MeSH
Related in: MedlinePlus