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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 Pdcd4 down-regulates E-cadherin and activates β-catenin/Tcf dependent transcription in GEO cells. (a) Knock-down of Pdcd4 down-regulates E-cadherin. Western blot was performed using antibodies against E-cadherin, β-catenin, and GAPDH. The ratios of E-cadherin/GAPDH and β-catenin/GAPDH in GEO cells are designated as 1.0. The number indicates the relative level of E-cadherin or β-catenin protein. (b) Knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription. GEO-shLacZ and GEO-shPdcd4 cells were transfected with either 0.2 μg of TOPflash or FOPflash along with 10 ng of pRL-SV40. TOPflash and FOPflash constructs are widely used to evaluate β-catenin/Tcf dependent signaling events that regulated by TCF/LEF family. TOPflash construct driven by thymidine kinase minimal promoter contains 6 wild-type Tcf binding sites upstream of a luciferase reporter gene. FOPflash is similar as TOPflash except that it contains 6 mutated Tcf binding sites. FOPflash is used as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity, and the activity of GEO-shLacZ cells transfected with either TOPflash or FOPflash plasmid is designated as 1. These experiments were repeated three times, each with five independent transfections; representative data are shown. Results are expressed as mean ± standard deviation (SD). The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with TOPflash, as determined by one-way ANOVA (p < 0.0001).
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Figure 1: Knock-down of Pdcd4 down-regulates E-cadherin and activates β-catenin/Tcf dependent transcription in GEO cells. (a) Knock-down of Pdcd4 down-regulates E-cadherin. Western blot was performed using antibodies against E-cadherin, β-catenin, and GAPDH. The ratios of E-cadherin/GAPDH and β-catenin/GAPDH in GEO cells are designated as 1.0. The number indicates the relative level of E-cadherin or β-catenin protein. (b) Knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription. GEO-shLacZ and GEO-shPdcd4 cells were transfected with either 0.2 μg of TOPflash or FOPflash along with 10 ng of pRL-SV40. TOPflash and FOPflash constructs are widely used to evaluate β-catenin/Tcf dependent signaling events that regulated by TCF/LEF family. TOPflash construct driven by thymidine kinase minimal promoter contains 6 wild-type Tcf binding sites upstream of a luciferase reporter gene. FOPflash is similar as TOPflash except that it contains 6 mutated Tcf binding sites. FOPflash is used as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity, and the activity of GEO-shLacZ cells transfected with either TOPflash or FOPflash plasmid is designated as 1. These experiments were repeated three times, each with five independent transfections; representative data are shown. Results are expressed as mean ± standard deviation (SD). The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with TOPflash, as determined by one-way ANOVA (p < 0.0001).

Mentions: Previously, we have demonstrated that Pdcd4 knock-down in HT29 cells resulted in down-regulation of E-cadherin and a slight decrease in β-catenin protein level (Wang et al., 2008). Although Pdcd4 knock-down in GEO cells also down-regulated E-cadherin expression, the protein level of β-catenin decreased dramatically with more than 70% relative to GEO-shLacZ cells (Figure 1a). The GEO-shLacZ and parental GEO cells had similar levels of β-catenin protein (Figure 1a). This finding thus raises a question as to whether Pdcd4 knock-down is able to activate β-catenin/Tcf dependent transcription in GEO cells. To address this question, we transfected TOPflash and FOPflash luciferase reporter constructs into GEO-shLacZ and GEO-shPdcd4 cells. The TOPflash and FOPflash contain the wild type and mutated β-catenin/Tcf binding site, respectively. As shown in Figure 1b, β-catenin/Tcf dependent transcription was approximately 3-fold-higher in GEO-shPdcd4 cells relative to GEO-shLacZ cells (filled bars). To confirm the specificity of this induction, the FOPflash plasmid was transfected into GEO-shLacZ and GEO-shPdcd4 cells. There was no significant difference in luciferase activity between the GEO-shLacZ and GEO-shPdcd4 cells (Figure 1b, open bars). In addition, we found that knock-down of Pdcd4 in GEO cells also led to translocation of β-catenin into nucleus (Supplementary Figure S1) and promotion of invasion (Supplementary Figure S2). These data clearly show that knock-down of Pdcd4 expression activates β-catenin/Tcf dependent transcription and promotes invasion in GEO cells. The extent of activation of β-catenin/Tcf dependent transcription in GEO-shPdcd4 cells is lower compared to that of the HT29-shPdcd4 cells (Wang et al., 2008); this might be due to reduced accumulation of β-catenin in the nuclei of GEO-shPdcd4 cells as a consequence of decreased β-catenin protein level.


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 Pdcd4 down-regulates E-cadherin and activates β-catenin/Tcf dependent transcription in GEO cells. (a) Knock-down of Pdcd4 down-regulates E-cadherin. Western blot was performed using antibodies against E-cadherin, β-catenin, and GAPDH. The ratios of E-cadherin/GAPDH and β-catenin/GAPDH in GEO cells are designated as 1.0. The number indicates the relative level of E-cadherin or β-catenin protein. (b) Knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription. GEO-shLacZ and GEO-shPdcd4 cells were transfected with either 0.2 μg of TOPflash or FOPflash along with 10 ng of pRL-SV40. TOPflash and FOPflash constructs are widely used to evaluate β-catenin/Tcf dependent signaling events that regulated by TCF/LEF family. TOPflash construct driven by thymidine kinase minimal promoter contains 6 wild-type Tcf binding sites upstream of a luciferase reporter gene. FOPflash is similar as TOPflash except that it contains 6 mutated Tcf binding sites. FOPflash is used as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity, and the activity of GEO-shLacZ cells transfected with either TOPflash or FOPflash plasmid is designated as 1. These experiments were repeated three times, each with five independent transfections; representative data are shown. Results are expressed as mean ± standard deviation (SD). The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with TOPflash, as determined by one-way ANOVA (p < 0.0001).
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Related In: Results  -  Collection

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Figure 1: Knock-down of Pdcd4 down-regulates E-cadherin and activates β-catenin/Tcf dependent transcription in GEO cells. (a) Knock-down of Pdcd4 down-regulates E-cadherin. Western blot was performed using antibodies against E-cadherin, β-catenin, and GAPDH. The ratios of E-cadherin/GAPDH and β-catenin/GAPDH in GEO cells are designated as 1.0. The number indicates the relative level of E-cadherin or β-catenin protein. (b) Knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription. GEO-shLacZ and GEO-shPdcd4 cells were transfected with either 0.2 μg of TOPflash or FOPflash along with 10 ng of pRL-SV40. TOPflash and FOPflash constructs are widely used to evaluate β-catenin/Tcf dependent signaling events that regulated by TCF/LEF family. TOPflash construct driven by thymidine kinase minimal promoter contains 6 wild-type Tcf binding sites upstream of a luciferase reporter gene. FOPflash is similar as TOPflash except that it contains 6 mutated Tcf binding sites. FOPflash is used as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity, and the activity of GEO-shLacZ cells transfected with either TOPflash or FOPflash plasmid is designated as 1. These experiments were repeated three times, each with five independent transfections; representative data are shown. Results are expressed as mean ± standard deviation (SD). The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with TOPflash, as determined by one-way ANOVA (p < 0.0001).
Mentions: Previously, we have demonstrated that Pdcd4 knock-down in HT29 cells resulted in down-regulation of E-cadherin and a slight decrease in β-catenin protein level (Wang et al., 2008). Although Pdcd4 knock-down in GEO cells also down-regulated E-cadherin expression, the protein level of β-catenin decreased dramatically with more than 70% relative to GEO-shLacZ cells (Figure 1a). The GEO-shLacZ and parental GEO cells had similar levels of β-catenin protein (Figure 1a). This finding thus raises a question as to whether Pdcd4 knock-down is able to activate β-catenin/Tcf dependent transcription in GEO cells. To address this question, we transfected TOPflash and FOPflash luciferase reporter constructs into GEO-shLacZ and GEO-shPdcd4 cells. The TOPflash and FOPflash contain the wild type and mutated β-catenin/Tcf binding site, respectively. As shown in Figure 1b, β-catenin/Tcf dependent transcription was approximately 3-fold-higher in GEO-shPdcd4 cells relative to GEO-shLacZ cells (filled bars). To confirm the specificity of this induction, the FOPflash plasmid was transfected into GEO-shLacZ and GEO-shPdcd4 cells. There was no significant difference in luciferase activity between the GEO-shLacZ and GEO-shPdcd4 cells (Figure 1b, open bars). In addition, we found that knock-down of Pdcd4 in GEO cells also led to translocation of β-catenin into nucleus (Supplementary Figure S1) and promotion of invasion (Supplementary Figure S2). These data clearly show that knock-down of Pdcd4 expression activates β-catenin/Tcf dependent transcription and promotes invasion in GEO cells. The extent of activation of β-catenin/Tcf dependent transcription in GEO-shPdcd4 cells is lower compared to that of the HT29-shPdcd4 cells (Wang et al., 2008); this might be due to reduced accumulation of β-catenin in the nuclei of GEO-shPdcd4 cells as a consequence of decreased β-catenin protein level.

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