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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 3. TGFβ regulates c-Jun in a p38-dependent manner in PC-3U cells. (A and B) Cell lysates derived from PC-3U cells, treated or transfected as indicated, were subjected to immunoblotting for p-Ser63-c-Jun, total c-Jun, p-p38, p-GSK-3β Ser9, total GSK-3β and HA (p38). Actin served as internal control. (C) PC-3U cells transiently transfected with HA-p38 wild-type or HA-p38DN were subjected to a non-radioactive in vitro kinase assay using recombinant c-Jun, and cell lysates were subsequently subjected to immunoprecipitation (IP) with HA-antibodies. The product was immunoblotted with p-Ser63-c-Jun antibodies. (D) Inhibitor of p38 (SB203580) were added 1 h before treatment of cells. (E) PC-3U cells transiently transfected with wild-type or Ser9Ala mutant GSK-3β were treated with or without TGFβ, as indicated. The cells were then lysed and subjected to immunoblotting using p-Ser63-c-Jun and GSK-3β antisera. β-tubulin served as internal control for equal loading of proteins.
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Figure 3: Figure 3. TGFβ regulates c-Jun in a p38-dependent manner in PC-3U cells. (A and B) Cell lysates derived from PC-3U cells, treated or transfected as indicated, were subjected to immunoblotting for p-Ser63-c-Jun, total c-Jun, p-p38, p-GSK-3β Ser9, total GSK-3β and HA (p38). Actin served as internal control. (C) PC-3U cells transiently transfected with HA-p38 wild-type or HA-p38DN were subjected to a non-radioactive in vitro kinase assay using recombinant c-Jun, and cell lysates were subsequently subjected to immunoprecipitation (IP) with HA-antibodies. The product was immunoblotted with p-Ser63-c-Jun antibodies. (D) Inhibitor of p38 (SB203580) were added 1 h before treatment of cells. (E) PC-3U cells transiently transfected with wild-type or Ser9Ala mutant GSK-3β were treated with or without TGFβ, as indicated. The cells were then lysed and subjected to immunoblotting using p-Ser63-c-Jun and GSK-3β antisera. β-tubulin served as internal control for equal loading of proteins.

Mentions: To investigate if the TRAF6-induced effects on p21 and c-Jun expression, involves the p38 MAPK pathway, the p38 inhibitor SB203580 was used. In the presence of the p38 inhibitor the TGFβ-induced phosphorylation of c-Jun was suppressed, whereas the phosphorylation of JNK was unaltered (Fig. 3A). Osmotic shock was used as a positive control to detect p-JNK.


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 3. TGFβ regulates c-Jun in a p38-dependent manner in PC-3U cells. (A and B) Cell lysates derived from PC-3U cells, treated or transfected as indicated, were subjected to immunoblotting for p-Ser63-c-Jun, total c-Jun, p-p38, p-GSK-3β Ser9, total GSK-3β and HA (p38). Actin served as internal control. (C) PC-3U cells transiently transfected with HA-p38 wild-type or HA-p38DN were subjected to a non-radioactive in vitro kinase assay using recombinant c-Jun, and cell lysates were subsequently subjected to immunoprecipitation (IP) with HA-antibodies. The product was immunoblotted with p-Ser63-c-Jun antibodies. (D) Inhibitor of p38 (SB203580) were added 1 h before treatment of cells. (E) PC-3U cells transiently transfected with wild-type or Ser9Ala mutant GSK-3β were treated with or without TGFβ, as indicated. The cells were then lysed and subjected to immunoblotting using p-Ser63-c-Jun and GSK-3β antisera. β-tubulin served as internal control for equal loading of proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Figure 3. TGFβ regulates c-Jun in a p38-dependent manner in PC-3U cells. (A and B) Cell lysates derived from PC-3U cells, treated or transfected as indicated, were subjected to immunoblotting for p-Ser63-c-Jun, total c-Jun, p-p38, p-GSK-3β Ser9, total GSK-3β and HA (p38). Actin served as internal control. (C) PC-3U cells transiently transfected with HA-p38 wild-type or HA-p38DN were subjected to a non-radioactive in vitro kinase assay using recombinant c-Jun, and cell lysates were subsequently subjected to immunoprecipitation (IP) with HA-antibodies. The product was immunoblotted with p-Ser63-c-Jun antibodies. (D) Inhibitor of p38 (SB203580) were added 1 h before treatment of cells. (E) PC-3U cells transiently transfected with wild-type or Ser9Ala mutant GSK-3β were treated with or without TGFβ, as indicated. The cells were then lysed and subjected to immunoblotting using p-Ser63-c-Jun and GSK-3β antisera. β-tubulin served as internal control for equal loading of proteins.
Mentions: To investigate if the TRAF6-induced effects on p21 and c-Jun expression, involves the p38 MAPK pathway, the p38 inhibitor SB203580 was used. In the presence of the p38 inhibitor the TGFβ-induced phosphorylation of c-Jun was suppressed, whereas the phosphorylation of JNK was unaltered (Fig. 3A). Osmotic shock was used as a positive control to detect p-JNK.

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