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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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E-cadherin modulates β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. (a) Knock-down of E-cadherin activates β-catenin/Tcf dependent transcription in HT29 cells. One microgram of either E-cadherin shRNA (pRS-shEcad) or control shRNA (pRS-shGFP) was transfected into HT29 cells along with either 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pRS-shGFP plasmid is designated as 1.0. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001). (b) and (c) Over-expression of E-cadherin inhibits β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. Increasing amounts (0-0.1 μg) of pCMV6-Ecad plasmid were transfected into GEO-shPdcd4 cells with either 0.2 μg of TOPflash (b) or FOPflash (c) reporter as described in (a). The total DNA for each transfection was maintained at 0.3 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with either TOPflash or FOPflash but without pCMV6-Ecad plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected 0.1 μg of pCMV6-Ecad plasmid, as determined by one-way ANOVA (p < 0.005). (d) β-Galactosidase does not inhibit β-catenin/Tcf dependent transcription. One tenth microgram of pcDNA3.1, pCMV6-Ecad or pSV-β-gal plasmid was transfected into GEO-shPdcd4 cells with 0.2 μg of TOPflash plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of the cells transfected with pcDNA3.1 is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with pcDNA3.1, as determined by one-way ANOVA (p < 0.001). These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD.
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Figure 4: E-cadherin modulates β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. (a) Knock-down of E-cadherin activates β-catenin/Tcf dependent transcription in HT29 cells. One microgram of either E-cadherin shRNA (pRS-shEcad) or control shRNA (pRS-shGFP) was transfected into HT29 cells along with either 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pRS-shGFP plasmid is designated as 1.0. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001). (b) and (c) Over-expression of E-cadherin inhibits β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. Increasing amounts (0-0.1 μg) of pCMV6-Ecad plasmid were transfected into GEO-shPdcd4 cells with either 0.2 μg of TOPflash (b) or FOPflash (c) reporter as described in (a). The total DNA for each transfection was maintained at 0.3 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with either TOPflash or FOPflash but without pCMV6-Ecad plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected 0.1 μg of pCMV6-Ecad plasmid, as determined by one-way ANOVA (p < 0.005). (d) β-Galactosidase does not inhibit β-catenin/Tcf dependent transcription. One tenth microgram of pcDNA3.1, pCMV6-Ecad or pSV-β-gal plasmid was transfected into GEO-shPdcd4 cells with 0.2 μg of TOPflash plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of the cells transfected with pcDNA3.1 is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with pcDNA3.1, as determined by one-way ANOVA (p < 0.001). These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD.

Mentions: We demonstrated herein that Pdcd4 knock-down led to E-cadherin down-regulation and activation of β-catenin/Tcf dependent transcription (Figure 1). However, it is unclear whether down-regulation of E-cadherin is necessary for activating β-catenin/Tcf dependent transcription when Pdcd4 is knocked down. To address this issue, E-cadherin shRNA expression plasmid (pRS-shEcad) was transiently transfected into HT29 cells along with either TOPflash or FOPflash construct. Knock-down of Pdcd4 in GEO and HT29 cells results in similar changes in cell morphology as well as the cellular functions including down-regulation of E-cadherin (Figures 1 and Supplementary Figure S2) (Wang et al., 2008). Thus, these two cell lines are interchangeable for the experiment of knocking-down E-cadherin. Since the transfection efficiency is very low for GEO cells, HT29 cells were used in the experiment of transiently knocking down E-cadherin. The luciferase activity in cells transfected with pRS-shEcad and TOPflash was approximately 2-fold higher than that in cells transfected with pRS-shGFP (a plasmid expressing GFP shRNA) and TOPflash (Figure 4a, filled bars). In contrast, co-transfection of pRS-shEcad or pRS-shGFP with FOPflash showed no significant difference in β-catenin/Tcf dependent transcription activity (Figure 4a, open bars), indicating that knocking down E-cadherin specifically activates β-catenin/Tcf dependent transcription rather than activates transcription in general.


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)

E-cadherin modulates β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. (a) Knock-down of E-cadherin activates β-catenin/Tcf dependent transcription in HT29 cells. One microgram of either E-cadherin shRNA (pRS-shEcad) or control shRNA (pRS-shGFP) was transfected into HT29 cells along with either 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pRS-shGFP plasmid is designated as 1.0. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001). (b) and (c) Over-expression of E-cadherin inhibits β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. Increasing amounts (0-0.1 μg) of pCMV6-Ecad plasmid were transfected into GEO-shPdcd4 cells with either 0.2 μg of TOPflash (b) or FOPflash (c) reporter as described in (a). The total DNA for each transfection was maintained at 0.3 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with either TOPflash or FOPflash but without pCMV6-Ecad plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected 0.1 μg of pCMV6-Ecad plasmid, as determined by one-way ANOVA (p < 0.005). (d) β-Galactosidase does not inhibit β-catenin/Tcf dependent transcription. One tenth microgram of pcDNA3.1, pCMV6-Ecad or pSV-β-gal plasmid was transfected into GEO-shPdcd4 cells with 0.2 μg of TOPflash plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of the cells transfected with pcDNA3.1 is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with pcDNA3.1, as determined by one-way ANOVA (p < 0.001). These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD.
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Figure 4: E-cadherin modulates β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. (a) Knock-down of E-cadherin activates β-catenin/Tcf dependent transcription in HT29 cells. One microgram of either E-cadherin shRNA (pRS-shEcad) or control shRNA (pRS-shGFP) was transfected into HT29 cells along with either 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pRS-shGFP plasmid is designated as 1.0. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001). (b) and (c) Over-expression of E-cadherin inhibits β-catenin/Tcf dependent transcription in Pdcd4 knock-down cells. Increasing amounts (0-0.1 μg) of pCMV6-Ecad plasmid were transfected into GEO-shPdcd4 cells with either 0.2 μg of TOPflash (b) or FOPflash (c) reporter as described in (a). The total DNA for each transfection was maintained at 0.3 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with either TOPflash or FOPflash but without pCMV6-Ecad plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected 0.1 μg of pCMV6-Ecad plasmid, as determined by one-way ANOVA (p < 0.005). (d) β-Galactosidase does not inhibit β-catenin/Tcf dependent transcription. One tenth microgram of pcDNA3.1, pCMV6-Ecad or pSV-β-gal plasmid was transfected into GEO-shPdcd4 cells with 0.2 μg of TOPflash plus 10 ng of pRL-SV40. The luciferase activity was normalized against Renilla activity, and the activity of the cells transfected with pcDNA3.1 is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with pcDNA3.1, as determined by one-way ANOVA (p < 0.001). These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD.
Mentions: We demonstrated herein that Pdcd4 knock-down led to E-cadherin down-regulation and activation of β-catenin/Tcf dependent transcription (Figure 1). However, it is unclear whether down-regulation of E-cadherin is necessary for activating β-catenin/Tcf dependent transcription when Pdcd4 is knocked down. To address this issue, E-cadherin shRNA expression plasmid (pRS-shEcad) was transiently transfected into HT29 cells along with either TOPflash or FOPflash construct. Knock-down of Pdcd4 in GEO and HT29 cells results in similar changes in cell morphology as well as the cellular functions including down-regulation of E-cadherin (Figures 1 and Supplementary Figure S2) (Wang et al., 2008). Thus, these two cell lines are interchangeable for the experiment of knocking-down E-cadherin. Since the transfection efficiency is very low for GEO cells, HT29 cells were used in the experiment of transiently knocking down E-cadherin. The luciferase activity in cells transfected with pRS-shEcad and TOPflash was approximately 2-fold higher than that in cells transfected with pRS-shGFP (a plasmid expressing GFP shRNA) and TOPflash (Figure 4a, filled bars). In contrast, co-transfection of pRS-shEcad or pRS-shGFP with FOPflash showed no significant difference in β-catenin/Tcf dependent transcription activity (Figure 4a, open bars), indicating that knocking down E-cadherin specifically activates β-catenin/Tcf dependent transcription rather than activates transcription in general.

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