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Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

Persad S, Troussard AA, McPhee TR, Mulholland DJ, Dedhar S - J. Cell Biol. (2001)

Bottom Line: We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN.TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity.Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

View Article: PubMed Central - PubMed

Affiliation: British Columbia Cancer Agency, Jack Bell Research Center, Vancouver V6H 3Z6, British Columbia, Canada.

ABSTRACT
beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

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PTEN and ILK-KD inhibit DNA binding activities of β-catenin–TCF complex. (A) Electrophoretic mobility shift assays, using an oligonucleotide containing a potential binding site for TCF, demonstrated an abundance of DNA–protein complex in empty vector–transfected PC3 cells (panel 1, lane 1). The quantities of the protein–DNA complex are reduced significantly in PC3 cells transfected with ILK-KD or PTEN (panel 1, lanes 2 and 3). Electrophoretic mobility shift assays performed in the presence of a supershifting antibody (anti–β-catenin antibody) confirmed that the transcription factor complex binding to the oligonucleotide includes β-catenin (panel 2). (B) Coimmunoprecipitation of TCF-4 or Lef-1 with β-catenin using nuclear lysates from empty vector (control), ILK-KD–, or PTEN-WT–transfected PC3 cells show reduced complex formation in ILK-KD– and PTEN-WT–transfected cells compared with control cells. (C) The relative β-catenin/TCF activities (TOPFLASH/FOPFLASH luciferase reporter) in response to the various components of the PI-3 kinase/PTEN pathway. PC3 cells were transiently transfected with 0.5 μg of TOPFLASH reporter together with 2.5 μg of empty vector, ILK-WT, ILK-KD, PTEN-WT, PKB-AAA, or GSK-3-WT. β-Catenin/TCF activity was dramatically reduced due to PTEN, ILK-KD, and GSK-3-WT. The effect of PKB-AAA upon TOPFLASH activity was more modest in comparison. Parallel cotransfections with the various plasmids and FOPFLASH served as a negative control for TOPFLASH activity.
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Figure 3: PTEN and ILK-KD inhibit DNA binding activities of β-catenin–TCF complex. (A) Electrophoretic mobility shift assays, using an oligonucleotide containing a potential binding site for TCF, demonstrated an abundance of DNA–protein complex in empty vector–transfected PC3 cells (panel 1, lane 1). The quantities of the protein–DNA complex are reduced significantly in PC3 cells transfected with ILK-KD or PTEN (panel 1, lanes 2 and 3). Electrophoretic mobility shift assays performed in the presence of a supershifting antibody (anti–β-catenin antibody) confirmed that the transcription factor complex binding to the oligonucleotide includes β-catenin (panel 2). (B) Coimmunoprecipitation of TCF-4 or Lef-1 with β-catenin using nuclear lysates from empty vector (control), ILK-KD–, or PTEN-WT–transfected PC3 cells show reduced complex formation in ILK-KD– and PTEN-WT–transfected cells compared with control cells. (C) The relative β-catenin/TCF activities (TOPFLASH/FOPFLASH luciferase reporter) in response to the various components of the PI-3 kinase/PTEN pathway. PC3 cells were transiently transfected with 0.5 μg of TOPFLASH reporter together with 2.5 μg of empty vector, ILK-WT, ILK-KD, PTEN-WT, PKB-AAA, or GSK-3-WT. β-Catenin/TCF activity was dramatically reduced due to PTEN, ILK-KD, and GSK-3-WT. The effect of PKB-AAA upon TOPFLASH activity was more modest in comparison. Parallel cotransfections with the various plasmids and FOPFLASH served as a negative control for TOPFLASH activity.

Mentions: It is known that β-catenin interacts with transcription factors of the TCF/LEF family and subsequently activates genes that are responsive to TCF/LEF family members (Giese et al. 1995; Behrens et al. 1996; Clevers and Grosschedl 1996; Molenaar et al. 1996; van de Wetering et al. 1997). Therefore, we analyzed the ability of β-catenin to form complexes with a TCF-4 consensus oligonucleotide in the presence of either PTEN-WT or dominant negative ILK. PC3 cells were transfected with empty vector, PTEN-WT, or ILK-KD, and nuclear extracts for gel mobility shift assays were prepared. The gel mobility shift assay demonstrated the presence of significant levels of protein–DNA complex in PC3 cells transfected with empty vector (Fig. 3 A, panel 1). However, the level of the protein–DNA complex was significantly reduced in PC3 cells transfected with either PTEN-WT or ILK-KD (lanes 2 and 3) compared with cells transfected with empty vector (lane 1).


Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

Persad S, Troussard AA, McPhee TR, Mulholland DJ, Dedhar S - J. Cell Biol. (2001)

PTEN and ILK-KD inhibit DNA binding activities of β-catenin–TCF complex. (A) Electrophoretic mobility shift assays, using an oligonucleotide containing a potential binding site for TCF, demonstrated an abundance of DNA–protein complex in empty vector–transfected PC3 cells (panel 1, lane 1). The quantities of the protein–DNA complex are reduced significantly in PC3 cells transfected with ILK-KD or PTEN (panel 1, lanes 2 and 3). Electrophoretic mobility shift assays performed in the presence of a supershifting antibody (anti–β-catenin antibody) confirmed that the transcription factor complex binding to the oligonucleotide includes β-catenin (panel 2). (B) Coimmunoprecipitation of TCF-4 or Lef-1 with β-catenin using nuclear lysates from empty vector (control), ILK-KD–, or PTEN-WT–transfected PC3 cells show reduced complex formation in ILK-KD– and PTEN-WT–transfected cells compared with control cells. (C) The relative β-catenin/TCF activities (TOPFLASH/FOPFLASH luciferase reporter) in response to the various components of the PI-3 kinase/PTEN pathway. PC3 cells were transiently transfected with 0.5 μg of TOPFLASH reporter together with 2.5 μg of empty vector, ILK-WT, ILK-KD, PTEN-WT, PKB-AAA, or GSK-3-WT. β-Catenin/TCF activity was dramatically reduced due to PTEN, ILK-KD, and GSK-3-WT. The effect of PKB-AAA upon TOPFLASH activity was more modest in comparison. Parallel cotransfections with the various plasmids and FOPFLASH served as a negative control for TOPFLASH activity.
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Related In: Results  -  Collection

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Figure 3: PTEN and ILK-KD inhibit DNA binding activities of β-catenin–TCF complex. (A) Electrophoretic mobility shift assays, using an oligonucleotide containing a potential binding site for TCF, demonstrated an abundance of DNA–protein complex in empty vector–transfected PC3 cells (panel 1, lane 1). The quantities of the protein–DNA complex are reduced significantly in PC3 cells transfected with ILK-KD or PTEN (panel 1, lanes 2 and 3). Electrophoretic mobility shift assays performed in the presence of a supershifting antibody (anti–β-catenin antibody) confirmed that the transcription factor complex binding to the oligonucleotide includes β-catenin (panel 2). (B) Coimmunoprecipitation of TCF-4 or Lef-1 with β-catenin using nuclear lysates from empty vector (control), ILK-KD–, or PTEN-WT–transfected PC3 cells show reduced complex formation in ILK-KD– and PTEN-WT–transfected cells compared with control cells. (C) The relative β-catenin/TCF activities (TOPFLASH/FOPFLASH luciferase reporter) in response to the various components of the PI-3 kinase/PTEN pathway. PC3 cells were transiently transfected with 0.5 μg of TOPFLASH reporter together with 2.5 μg of empty vector, ILK-WT, ILK-KD, PTEN-WT, PKB-AAA, or GSK-3-WT. β-Catenin/TCF activity was dramatically reduced due to PTEN, ILK-KD, and GSK-3-WT. The effect of PKB-AAA upon TOPFLASH activity was more modest in comparison. Parallel cotransfections with the various plasmids and FOPFLASH served as a negative control for TOPFLASH activity.
Mentions: It is known that β-catenin interacts with transcription factors of the TCF/LEF family and subsequently activates genes that are responsive to TCF/LEF family members (Giese et al. 1995; Behrens et al. 1996; Clevers and Grosschedl 1996; Molenaar et al. 1996; van de Wetering et al. 1997). Therefore, we analyzed the ability of β-catenin to form complexes with a TCF-4 consensus oligonucleotide in the presence of either PTEN-WT or dominant negative ILK. PC3 cells were transfected with empty vector, PTEN-WT, or ILK-KD, and nuclear extracts for gel mobility shift assays were prepared. The gel mobility shift assay demonstrated the presence of significant levels of protein–DNA complex in PC3 cells transfected with empty vector (Fig. 3 A, panel 1). However, the level of the protein–DNA complex was significantly reduced in PC3 cells transfected with either PTEN-WT or ILK-KD (lanes 2 and 3) compared with cells transfected with empty vector (lane 1).

Bottom Line: We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN.TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity.Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

View Article: PubMed Central - PubMed

Affiliation: British Columbia Cancer Agency, Jack Bell Research Center, Vancouver V6H 3Z6, British Columbia, Canada.

ABSTRACT
beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Show MeSH
Related in: MedlinePlus