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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 inhibits E-cadherin expression through elevated Snail expression. (a) Pdcd4 knock-down inhibits E-cadherin promoter activity. The E-cadherin promoter reporter constructs (0.2 μg), wild-type (E7) and E-box mutant (E7-m) were transfected into GEO-shLacZ and GEO-shPdcd4 cells along with 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity. The activity of GEO-shLacZ cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference as determined by one-way ANOVA (p< 0.005). (b) Snail expression is up-regulated in GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH as described in Materials and Methods. (c) Knock-down of Snail expression in GEO-shPdcd4 cells. The snail siRNA (si-snail) and control siRNA (control) was transfected into GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH. The ratios of Snail/GAPDH in control cells are designated as 1.0. The number indicates the relative level of Snail protein. (d) Knock-down of Snail in GEO-shPdcd4 cells activates E-cadherin promoter activity. The wild-type E7 promoter reporter construct (0.2 μg) along with 10 ng of pRL-SV40 were transfected into control and si-snail cells. The luciferase activity was normalized against Renilla activity. The activity of control cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with E7 as determined by one-way ANOVA (p < 0.05).
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Figure 2: Knock-down of Pdcd4 inhibits E-cadherin expression through elevated Snail expression. (a) Pdcd4 knock-down inhibits E-cadherin promoter activity. The E-cadherin promoter reporter constructs (0.2 μg), wild-type (E7) and E-box mutant (E7-m) were transfected into GEO-shLacZ and GEO-shPdcd4 cells along with 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity. The activity of GEO-shLacZ cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference as determined by one-way ANOVA (p< 0.005). (b) Snail expression is up-regulated in GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH as described in Materials and Methods. (c) Knock-down of Snail expression in GEO-shPdcd4 cells. The snail siRNA (si-snail) and control siRNA (control) was transfected into GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH. The ratios of Snail/GAPDH in control cells are designated as 1.0. The number indicates the relative level of Snail protein. (d) Knock-down of Snail in GEO-shPdcd4 cells activates E-cadherin promoter activity. The wild-type E7 promoter reporter construct (0.2 μg) along with 10 ng of pRL-SV40 were transfected into control and si-snail cells. The luciferase activity was normalized against Renilla activity. The activity of control cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with E7 as determined by one-way ANOVA (p < 0.05).

Mentions: To understand the mechanism by which Pdcd4 knock-down leads to down-regulation of E-cadherin expression, the E-cadherin promoter-luciferase plasmid (E7) was transfected into GEO-shLacZ and GEO-shPdcd4 cells. The E7 construct comprises E-cadherin promoter region from −38 to +135 bp (containing two E-box sites) fused with luciferase reporter (Liu et al., 2005). The luciferase activity in GEO-shPdcd4 cells was approximately 35% of that in GEO-shLacZ cells (Figure 2a), indicating that E-cadherin promoter activity is inhibited by Pdcd4 knock-down. To test whether the E-boxes mediate the inhibition of E-cadherin promoter activity in Pdcd4 knock-down cells, we mutated both E-box sites and transfected the mutated construct (E7-m) into GEO-shLacZ and GEO-shPdcd4 cells. The E7-m exhibited approximately 70% of the basal level of E-cadherin promoter activity (Figure 2a, filled bars). However, the luciferase activity transfected with E7-m construct was similar in GEO-shLacZ and GEO-shPdcd4 cells (Figure 2a, E7-m filled vs open bar), indicating that mutation of E-boxes abolished the inhibition of E-cadherin promoter activity in Pdcd4 knock-down cells. It has been known that transcription factor Snail can binds to E-boxes on the E-cadherin promoter and inhibits E-cadherin expression (Batlle et al., 2000; Cano et al., 2000). To determine whether Pdcd4 knock-down leads to the up-regulation of Snail expression, the protein level of Snail was examined. As shown in Figure 2b, the Snail protein level in GEO-shPdcd4 cells was dramatically induced while the Snail protein was undetectable in GEO-shLacZ cells. We then knocked down Snail expression and tested whether Snail knock-down can restore the E-cadherin promoter activity in GEO-shPdcd4 cells. The level of Snail protein in Snail knock-down cells (si-snail) was approximately 50% of that in GEO-shPdcd4 cells expressing control siRNA (control) (Figure 2c) whereas the E-cadherin promoter activity in Snail knock-down cells was approximately 180% of that in control cells (Figure 2d). These results indicate that Snail contributes to the inhibition of E-cadherin expression in GEO-shPdcd4 cells. Our findings indicate that Pdcd4 knock-down leads to up-regulation of Snail resulting in inhibition of E-cadherin expression.


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 inhibits E-cadherin expression through elevated Snail expression. (a) Pdcd4 knock-down inhibits E-cadherin promoter activity. The E-cadherin promoter reporter constructs (0.2 μg), wild-type (E7) and E-box mutant (E7-m) were transfected into GEO-shLacZ and GEO-shPdcd4 cells along with 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity. The activity of GEO-shLacZ cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference as determined by one-way ANOVA (p< 0.005). (b) Snail expression is up-regulated in GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH as described in Materials and Methods. (c) Knock-down of Snail expression in GEO-shPdcd4 cells. The snail siRNA (si-snail) and control siRNA (control) was transfected into GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH. The ratios of Snail/GAPDH in control cells are designated as 1.0. The number indicates the relative level of Snail protein. (d) Knock-down of Snail in GEO-shPdcd4 cells activates E-cadherin promoter activity. The wild-type E7 promoter reporter construct (0.2 μg) along with 10 ng of pRL-SV40 were transfected into control and si-snail cells. The luciferase activity was normalized against Renilla activity. The activity of control cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with E7 as determined by one-way ANOVA (p < 0.05).
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Figure 2: Knock-down of Pdcd4 inhibits E-cadherin expression through elevated Snail expression. (a) Pdcd4 knock-down inhibits E-cadherin promoter activity. The E-cadherin promoter reporter constructs (0.2 μg), wild-type (E7) and E-box mutant (E7-m) were transfected into GEO-shLacZ and GEO-shPdcd4 cells along with 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity. The activity of GEO-shLacZ cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference as determined by one-way ANOVA (p< 0.005). (b) Snail expression is up-regulated in GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH as described in Materials and Methods. (c) Knock-down of Snail expression in GEO-shPdcd4 cells. The snail siRNA (si-snail) and control siRNA (control) was transfected into GEO-shPdcd4 cells. Western blot was performed using antibodies against Snail and GAPDH. The ratios of Snail/GAPDH in control cells are designated as 1.0. The number indicates the relative level of Snail protein. (d) Knock-down of Snail in GEO-shPdcd4 cells activates E-cadherin promoter activity. The wild-type E7 promoter reporter construct (0.2 μg) along with 10 ng of pRL-SV40 were transfected into control and si-snail cells. The luciferase activity was normalized against Renilla activity. The activity of control cells transfected with E7 is designated as 100%. The asterisk denotes a significant difference compared with GEO-shLacZ cells transfected with E7 as determined by one-way ANOVA (p < 0.05).
Mentions: To understand the mechanism by which Pdcd4 knock-down leads to down-regulation of E-cadherin expression, the E-cadherin promoter-luciferase plasmid (E7) was transfected into GEO-shLacZ and GEO-shPdcd4 cells. The E7 construct comprises E-cadherin promoter region from −38 to +135 bp (containing two E-box sites) fused with luciferase reporter (Liu et al., 2005). The luciferase activity in GEO-shPdcd4 cells was approximately 35% of that in GEO-shLacZ cells (Figure 2a), indicating that E-cadherin promoter activity is inhibited by Pdcd4 knock-down. To test whether the E-boxes mediate the inhibition of E-cadherin promoter activity in Pdcd4 knock-down cells, we mutated both E-box sites and transfected the mutated construct (E7-m) into GEO-shLacZ and GEO-shPdcd4 cells. The E7-m exhibited approximately 70% of the basal level of E-cadherin promoter activity (Figure 2a, filled bars). However, the luciferase activity transfected with E7-m construct was similar in GEO-shLacZ and GEO-shPdcd4 cells (Figure 2a, E7-m filled vs open bar), indicating that mutation of E-boxes abolished the inhibition of E-cadherin promoter activity in Pdcd4 knock-down cells. It has been known that transcription factor Snail can binds to E-boxes on the E-cadherin promoter and inhibits E-cadherin expression (Batlle et al., 2000; Cano et al., 2000). To determine whether Pdcd4 knock-down leads to the up-regulation of Snail expression, the protein level of Snail was examined. As shown in Figure 2b, the Snail protein level in GEO-shPdcd4 cells was dramatically induced while the Snail protein was undetectable in GEO-shLacZ cells. We then knocked down Snail expression and tested whether Snail knock-down can restore the E-cadherin promoter activity in GEO-shPdcd4 cells. The level of Snail protein in Snail knock-down cells (si-snail) was approximately 50% of that in GEO-shPdcd4 cells expressing control siRNA (control) (Figure 2c) whereas the E-cadherin promoter activity in Snail knock-down cells was approximately 180% of that in control cells (Figure 2d). These results indicate that Snail contributes to the inhibition of E-cadherin expression in GEO-shPdcd4 cells. Our findings indicate that Pdcd4 knock-down leads to up-regulation of Snail resulting in inhibition of E-cadherin expression.

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