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TGFβ-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1.

Thakur N, Gudey SK, Marcusson A, Fu JY, Bergh A, Heldin CH, Landström M - Cell Cycle (2014)

Bottom Line: In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63.The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9.Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: a Ludwig Institute for Cancer Research; Science for Life Laboratory; Uppsala University; Uppsala, Sweden.

ABSTRACT
High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFβ conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TβRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFβ also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TβRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFβ that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

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Figure 11. c-Jun promotes TGFβ-induced invasion of prostate cancer cells. (A) Knock-down of c-Jun in PC-3U cells significantly prevented TGFβ-induced invasion. Data are presented as mean values (± S.D.) for invasive cells in 3 independent experiments (***, P < 0.001, when compared with control siRNA). (B) Invasion assay was performed in PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated. Cells were then visualized by staining with crystal violet cell stain solution. (C and D) Invasion assay was performed in PC-3U cells transiently transfected with CTRL, c-Jun siRNA alone or together with HA-tagged Snail1, and treated as indicated. (E) Cell lysates derived from a part of the cells in (C) was subjected to immunoblotting for total c-Jun and HA (Snail). Actin served as internal control for equal loading of proteins. (F) The number of proliferating PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated, was subjected to immunofluorescence stainings of phospo-Histone3 (p-H3). Data are presented as mean values (± S.D.) in 3 independent experiments. (**, P < 0.01, ***, P < 0.001, when compared with control siRNA).
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Figure 11: Figure 11. c-Jun promotes TGFβ-induced invasion of prostate cancer cells. (A) Knock-down of c-Jun in PC-3U cells significantly prevented TGFβ-induced invasion. Data are presented as mean values (± S.D.) for invasive cells in 3 independent experiments (***, P < 0.001, when compared with control siRNA). (B) Invasion assay was performed in PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated. Cells were then visualized by staining with crystal violet cell stain solution. (C and D) Invasion assay was performed in PC-3U cells transiently transfected with CTRL, c-Jun siRNA alone or together with HA-tagged Snail1, and treated as indicated. (E) Cell lysates derived from a part of the cells in (C) was subjected to immunoblotting for total c-Jun and HA (Snail). Actin served as internal control for equal loading of proteins. (F) The number of proliferating PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated, was subjected to immunofluorescence stainings of phospo-Histone3 (p-H3). Data are presented as mean values (± S.D.) in 3 independent experiments. (**, P < 0.01, ***, P < 0.001, when compared with control siRNA).

Mentions: Both p38 and c-Jun have been implicated in cell migration during embryogenesis and wound healing.7,37,39,41 To investigate the possible role of activated c-Jun in TGFβ-induced migration of PC-3U cells, we used siRNA to knock-down c-Jun or TRAF6. Silencing of c-Jun or TRAF6 caused a complete inhibition of the TGFβ-induced cell culture wound healing of PC-3U cells (Fig. 10A and B). Importantly, rescue experiments, in which c-Jun was transiently overexpressed in PC-3U cells with TRAF6 silenced, showed an increased cell migration (Fig. 10C and D). Moreover, c-Jun was found to be required for TGFβ-induced invasion of PC-3U cells through Matrigel (Fig. 11A and B). Ectopic expression of HA-tagged Snail1, partially rescued the loss of TGFβ-induced invasive properties in PC-3U cells when endogenous c-Jun was knocked down by siRNA, demonstrating that Snail1acts downstream of c-Jun, to promote invasion (Fig. 11C–E). TGFβ-induced growth inhibition in PC-3U cells and knock-down of c-Jun reduced the basal proliferative response in PC-3U cells (Fig. 11F). We noticed that silencing of c-Jun also reduced both the basal migratory capability and the invasiveness of PC-3U cells. These effects may be explained by the fact that PC-3U cells are known to secrete TGFβ2 in an autocrine fashion.42 From these observations we conclude that the TGFβ-induced invasive properties of PC-3U cells are conferred by activation of c-Jun, causing induction of Snail1.


TGFβ-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1.

Thakur N, Gudey SK, Marcusson A, Fu JY, Bergh A, Heldin CH, Landström M - Cell Cycle (2014)

Figure 11. c-Jun promotes TGFβ-induced invasion of prostate cancer cells. (A) Knock-down of c-Jun in PC-3U cells significantly prevented TGFβ-induced invasion. Data are presented as mean values (± S.D.) for invasive cells in 3 independent experiments (***, P < 0.001, when compared with control siRNA). (B) Invasion assay was performed in PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated. Cells were then visualized by staining with crystal violet cell stain solution. (C and D) Invasion assay was performed in PC-3U cells transiently transfected with CTRL, c-Jun siRNA alone or together with HA-tagged Snail1, and treated as indicated. (E) Cell lysates derived from a part of the cells in (C) was subjected to immunoblotting for total c-Jun and HA (Snail). Actin served as internal control for equal loading of proteins. (F) The number of proliferating PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated, was subjected to immunofluorescence stainings of phospo-Histone3 (p-H3). Data are presented as mean values (± S.D.) in 3 independent experiments. (**, P < 0.01, ***, P < 0.001, when compared with control siRNA).
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Figure 11: Figure 11. c-Jun promotes TGFβ-induced invasion of prostate cancer cells. (A) Knock-down of c-Jun in PC-3U cells significantly prevented TGFβ-induced invasion. Data are presented as mean values (± S.D.) for invasive cells in 3 independent experiments (***, P < 0.001, when compared with control siRNA). (B) Invasion assay was performed in PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated. Cells were then visualized by staining with crystal violet cell stain solution. (C and D) Invasion assay was performed in PC-3U cells transiently transfected with CTRL, c-Jun siRNA alone or together with HA-tagged Snail1, and treated as indicated. (E) Cell lysates derived from a part of the cells in (C) was subjected to immunoblotting for total c-Jun and HA (Snail). Actin served as internal control for equal loading of proteins. (F) The number of proliferating PC-3U cells transiently transfected with control (CTRL) or c-Jun siRNA, and treated as indicated, was subjected to immunofluorescence stainings of phospo-Histone3 (p-H3). Data are presented as mean values (± S.D.) in 3 independent experiments. (**, P < 0.01, ***, P < 0.001, when compared with control siRNA).
Mentions: Both p38 and c-Jun have been implicated in cell migration during embryogenesis and wound healing.7,37,39,41 To investigate the possible role of activated c-Jun in TGFβ-induced migration of PC-3U cells, we used siRNA to knock-down c-Jun or TRAF6. Silencing of c-Jun or TRAF6 caused a complete inhibition of the TGFβ-induced cell culture wound healing of PC-3U cells (Fig. 10A and B). Importantly, rescue experiments, in which c-Jun was transiently overexpressed in PC-3U cells with TRAF6 silenced, showed an increased cell migration (Fig. 10C and D). Moreover, c-Jun was found to be required for TGFβ-induced invasion of PC-3U cells through Matrigel (Fig. 11A and B). Ectopic expression of HA-tagged Snail1, partially rescued the loss of TGFβ-induced invasive properties in PC-3U cells when endogenous c-Jun was knocked down by siRNA, demonstrating that Snail1acts downstream of c-Jun, to promote invasion (Fig. 11C–E). TGFβ-induced growth inhibition in PC-3U cells and knock-down of c-Jun reduced the basal proliferative response in PC-3U cells (Fig. 11F). We noticed that silencing of c-Jun also reduced both the basal migratory capability and the invasiveness of PC-3U cells. These effects may be explained by the fact that PC-3U cells are known to secrete TGFβ2 in an autocrine fashion.42 From these observations we conclude that the TGFβ-induced invasive properties of PC-3U cells are conferred by activation of c-Jun, causing induction of Snail1.

Bottom Line: In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63.The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9.Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: a Ludwig Institute for Cancer Research; Science for Life Laboratory; Uppsala University; Uppsala, Sweden.

ABSTRACT
High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFβ conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TβRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFβ also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TβRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFβ that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

Show MeSH
Related in: MedlinePlus