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Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

Wang Q, Sun ZX, Allgayer H, Yang HS - Oncogene (2009)

Bottom Line: In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells.Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells.Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

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Knock-down of u-PAR or c-Myc inhibits invasion in Pdcd4 knock-down cells. (a) Knock-down of u-PAR and c-Myc in GEO-shPdcd4 cells. The u-PAR and c-Myc expression was knocked down by transfection with u-PAR siRNA and c-myc siRNA, respectively. The u-PAR and c-Myc protein levels from control siRNA (control), u-PAR siRNA (si-u-PAR), and c-myc siRNA (si-Myc) expressing cells were determined by Western blot using antibodies against u-PAR, c-Myc, and GAPDH. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in control cells is designated as 1. The number indicates the relative protein level of u-PAR or c-Myc. (b) Knock-down of u-PAR and c-Myc inhibits invasion. Matrigel invasion assays were performed with the indicated cells for 48 h using FBS (0.5%) and EGF (20 ng/ml) as attractants. Each value is the mean ± SD of three independent experiments. The asterisk denotes a significant difference compared with control cells as determined by one-way ANOVA (P < 0.05).
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Figure 6: Knock-down of u-PAR or c-Myc inhibits invasion in Pdcd4 knock-down cells. (a) Knock-down of u-PAR and c-Myc in GEO-shPdcd4 cells. The u-PAR and c-Myc expression was knocked down by transfection with u-PAR siRNA and c-myc siRNA, respectively. The u-PAR and c-Myc protein levels from control siRNA (control), u-PAR siRNA (si-u-PAR), and c-myc siRNA (si-Myc) expressing cells were determined by Western blot using antibodies against u-PAR, c-Myc, and GAPDH. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in control cells is designated as 1. The number indicates the relative protein level of u-PAR or c-Myc. (b) Knock-down of u-PAR and c-Myc inhibits invasion. Matrigel invasion assays were performed with the indicated cells for 48 h using FBS (0.5%) and EGF (20 ng/ml) as attractants. Each value is the mean ± SD of three independent experiments. The asterisk denotes a significant difference compared with control cells as determined by one-way ANOVA (P < 0.05).

Mentions: To test whether elevated u-PAR and c-Myc expression contributes to the promotion of invasion in Pdcd4 knock-down cells, we knocked down u-PAR or c-Myc by transient transfection of u-PAR or c-myc siRNA into GEO-shPdcd4 cells. After 7 days, the u-PAR or c-Myc knock-down cells were then assayed for the capacity to pass through a Matrigel barrier in a modified Boyden chamber assay using FBS (0.5%) and EGF (20 ng/ml) as attractants. As shown in Figure 6a, u-PAR knock-down (si-u-PAR) and c-Myc knock-down (si-Myc) cells displayed about 50% and 80% reduction of u-PAR and c-Myc expression, respectively. The ability of si-u-PAR and si-Myc cells to invade through Matrigel was reduced to 50% and 30% of that in control cells, respectively (Figure 6b). These results indicate that both u-PAR and c-Myc contribute to the invasion induced by Pdcd4 knock-down, providing molecular explanation of how Pdcd4 suppresses tumor invasion.


Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

Wang Q, Sun ZX, Allgayer H, Yang HS - Oncogene (2009)

Knock-down of u-PAR or c-Myc inhibits invasion in Pdcd4 knock-down cells. (a) Knock-down of u-PAR and c-Myc in GEO-shPdcd4 cells. The u-PAR and c-Myc expression was knocked down by transfection with u-PAR siRNA and c-myc siRNA, respectively. The u-PAR and c-Myc protein levels from control siRNA (control), u-PAR siRNA (si-u-PAR), and c-myc siRNA (si-Myc) expressing cells were determined by Western blot using antibodies against u-PAR, c-Myc, and GAPDH. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in control cells is designated as 1. The number indicates the relative protein level of u-PAR or c-Myc. (b) Knock-down of u-PAR and c-Myc inhibits invasion. Matrigel invasion assays were performed with the indicated cells for 48 h using FBS (0.5%) and EGF (20 ng/ml) as attractants. Each value is the mean ± SD of three independent experiments. The asterisk denotes a significant difference compared with control cells as determined by one-way ANOVA (P < 0.05).
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Related In: Results  -  Collection

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Figure 6: Knock-down of u-PAR or c-Myc inhibits invasion in Pdcd4 knock-down cells. (a) Knock-down of u-PAR and c-Myc in GEO-shPdcd4 cells. The u-PAR and c-Myc expression was knocked down by transfection with u-PAR siRNA and c-myc siRNA, respectively. The u-PAR and c-Myc protein levels from control siRNA (control), u-PAR siRNA (si-u-PAR), and c-myc siRNA (si-Myc) expressing cells were determined by Western blot using antibodies against u-PAR, c-Myc, and GAPDH. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in control cells is designated as 1. The number indicates the relative protein level of u-PAR or c-Myc. (b) Knock-down of u-PAR and c-Myc inhibits invasion. Matrigel invasion assays were performed with the indicated cells for 48 h using FBS (0.5%) and EGF (20 ng/ml) as attractants. Each value is the mean ± SD of three independent experiments. The asterisk denotes a significant difference compared with control cells as determined by one-way ANOVA (P < 0.05).
Mentions: To test whether elevated u-PAR and c-Myc expression contributes to the promotion of invasion in Pdcd4 knock-down cells, we knocked down u-PAR or c-Myc by transient transfection of u-PAR or c-myc siRNA into GEO-shPdcd4 cells. After 7 days, the u-PAR or c-Myc knock-down cells were then assayed for the capacity to pass through a Matrigel barrier in a modified Boyden chamber assay using FBS (0.5%) and EGF (20 ng/ml) as attractants. As shown in Figure 6a, u-PAR knock-down (si-u-PAR) and c-Myc knock-down (si-Myc) cells displayed about 50% and 80% reduction of u-PAR and c-Myc expression, respectively. The ability of si-u-PAR and si-Myc cells to invade through Matrigel was reduced to 50% and 30% of that in control cells, respectively (Figure 6b). These results indicate that both u-PAR and c-Myc contribute to the invasion induced by Pdcd4 knock-down, providing molecular explanation of how Pdcd4 suppresses tumor invasion.

Bottom Line: In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells.Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells.Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

Show MeSH
Related in: MedlinePlus