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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 4. TGFβ regulates c-Jun and p21 in PC-3U cells. (A) PC-3U cells treated with or without TGFβ were subjected to nuclear and cytoplasmic fractionation; the fractions were then analyzed by immunoblotting with p-Ser63-c-Jun, total c-Jun and p-Smad2 antibodies. Beta-tubulin and the transcription factor YY1 served as controls for cytoplasmic and nuclear fractions, respectively. (B) Cell lysates derived from PC-3U cells were treated with TGFβ as indicated, and then subjected to immunoblotting for p21. The expression of p21 is shown in comparison with immunoblotting for p-Ser63-c-Jun, total c-Jun and p-Smad2. Actin served as internal control. (C and D) RNA derived from PC-3U cells treated with TGFβ for the indicated time periods, was subjected to qRT-PCR for c-Jun and p21.
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Figure 4: Figure 4. TGFβ regulates c-Jun and p21 in PC-3U cells. (A) PC-3U cells treated with or without TGFβ were subjected to nuclear and cytoplasmic fractionation; the fractions were then analyzed by immunoblotting with p-Ser63-c-Jun, total c-Jun and p-Smad2 antibodies. Beta-tubulin and the transcription factor YY1 served as controls for cytoplasmic and nuclear fractions, respectively. (B) Cell lysates derived from PC-3U cells were treated with TGFβ as indicated, and then subjected to immunoblotting for p21. The expression of p21 is shown in comparison with immunoblotting for p-Ser63-c-Jun, total c-Jun and p-Smad2. Actin served as internal control. (C and D) RNA derived from PC-3U cells treated with TGFβ for the indicated time periods, was subjected to qRT-PCR for c-Jun and p21.

Mentions: To analyze the subcellular localization of active c-Jun, nuclear and cytoplasmic fractions of cells were analyzed separately. Increased expression and phosphorylation of c-Jun, and phosphorylation of Smad2, were observed in the nuclear fraction, but not in the cytoplasmic fraction, after TGFβ stimulation (Fig. 4A). We further validated the expression patterns of c-Jun and p21 at the protein level. The expression of p21 and c-Jun was enhanced upon stimulation with TGFβ, whereas phosphorylation of Smad2 was enhanced up to 60 min and thereafter declined (Fig. 4B). Upon stimulation with TGFβ, the c-Jun mRNA expression in PC-3U cells increased until 120 min (Fig. 4C) and thereafter decreased, whereas the expression of p21 mRNA increased until 12 h (Fig. 4D).


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 4. TGFβ regulates c-Jun and p21 in PC-3U cells. (A) PC-3U cells treated with or without TGFβ were subjected to nuclear and cytoplasmic fractionation; the fractions were then analyzed by immunoblotting with p-Ser63-c-Jun, total c-Jun and p-Smad2 antibodies. Beta-tubulin and the transcription factor YY1 served as controls for cytoplasmic and nuclear fractions, respectively. (B) Cell lysates derived from PC-3U cells were treated with TGFβ as indicated, and then subjected to immunoblotting for p21. The expression of p21 is shown in comparison with immunoblotting for p-Ser63-c-Jun, total c-Jun and p-Smad2. Actin served as internal control. (C and D) RNA derived from PC-3U cells treated with TGFβ for the indicated time periods, was subjected to qRT-PCR for c-Jun and p21.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Figure 4. TGFβ regulates c-Jun and p21 in PC-3U cells. (A) PC-3U cells treated with or without TGFβ were subjected to nuclear and cytoplasmic fractionation; the fractions were then analyzed by immunoblotting with p-Ser63-c-Jun, total c-Jun and p-Smad2 antibodies. Beta-tubulin and the transcription factor YY1 served as controls for cytoplasmic and nuclear fractions, respectively. (B) Cell lysates derived from PC-3U cells were treated with TGFβ as indicated, and then subjected to immunoblotting for p21. The expression of p21 is shown in comparison with immunoblotting for p-Ser63-c-Jun, total c-Jun and p-Smad2. Actin served as internal control. (C and D) RNA derived from PC-3U cells treated with TGFβ for the indicated time periods, was subjected to qRT-PCR for c-Jun and p21.
Mentions: To analyze the subcellular localization of active c-Jun, nuclear and cytoplasmic fractions of cells were analyzed separately. Increased expression and phosphorylation of c-Jun, and phosphorylation of Smad2, were observed in the nuclear fraction, but not in the cytoplasmic fraction, after TGFβ stimulation (Fig. 4A). We further validated the expression patterns of c-Jun and p21 at the protein level. The expression of p21 and c-Jun was enhanced upon stimulation with TGFβ, whereas phosphorylation of Smad2 was enhanced up to 60 min and thereafter declined (Fig. 4B). Upon stimulation with TGFβ, the c-Jun mRNA expression in PC-3U cells increased until 120 min (Fig. 4C) and thereafter decreased, whereas the expression of p21 mRNA increased until 12 h (Fig. 4D).

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