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A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

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F-TrCP-Ecad promotes β-catenin degradation and inhibits Wnt signaling in DLD1 cells. A. Effects of F-TrCP-Ecad on the levels of cytosolic and membrane-associated β-catenin. A DLD1 inducible cell line was generated with the tetracycline (tet)-off system; these cells expressed F-TrCP-Ecad only in the absence of Dox. Cells were grown in the presence or absence of Dox for 5 days, and subjected to subcellular fractionation. Equal amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the anti-β-catenin antibody. α-tubulin levels were determined by immunoblotting with the anti-α-tubulin antibody as an internal loading control. Quantified representation of immunoblot is shown at the bottom of each blot (relative intensity). B. F-TrCP-Ecad inhibits the transcriptional activity of β-catenin. DLD1-F-TrCP-Ecad cells were transfected with TOP-FLASH and a CMV-Renilla reporter, and cultured in the presence or absence of Dox. The luciferase activity of cells grown in the presence of Dox was arbitrarily set to 1. C. F-TrCP-Ecad induction inhibits c-MYC expression. DLD1-F-TrCP-Ecad cells were cultured in the presence or absence of Dox for four days. The expression level of c-myc and F-TrCP-Ecad were determined by immunoblotting with the anti-MYC (C-19) and the anti-FLAG antibodies (top and middle panels). Equal loadings were confirmed by immunoblotting with the anti-α-tubulin antibody (bottom panel). 'o' indicates two unknown species immuno-reactive with the anti-FLAG antibody, which migrated right below βTrCP-Ecad of the middle panel.
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Figure 3: F-TrCP-Ecad promotes β-catenin degradation and inhibits Wnt signaling in DLD1 cells. A. Effects of F-TrCP-Ecad on the levels of cytosolic and membrane-associated β-catenin. A DLD1 inducible cell line was generated with the tetracycline (tet)-off system; these cells expressed F-TrCP-Ecad only in the absence of Dox. Cells were grown in the presence or absence of Dox for 5 days, and subjected to subcellular fractionation. Equal amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the anti-β-catenin antibody. α-tubulin levels were determined by immunoblotting with the anti-α-tubulin antibody as an internal loading control. Quantified representation of immunoblot is shown at the bottom of each blot (relative intensity). B. F-TrCP-Ecad inhibits the transcriptional activity of β-catenin. DLD1-F-TrCP-Ecad cells were transfected with TOP-FLASH and a CMV-Renilla reporter, and cultured in the presence or absence of Dox. The luciferase activity of cells grown in the presence of Dox was arbitrarily set to 1. C. F-TrCP-Ecad induction inhibits c-MYC expression. DLD1-F-TrCP-Ecad cells were cultured in the presence or absence of Dox for four days. The expression level of c-myc and F-TrCP-Ecad were determined by immunoblotting with the anti-MYC (C-19) and the anti-FLAG antibodies (top and middle panels). Equal loadings were confirmed by immunoblotting with the anti-α-tubulin antibody (bottom panel). 'o' indicates two unknown species immuno-reactive with the anti-FLAG antibody, which migrated right below βTrCP-Ecad of the middle panel.

Mentions: We tested the effect of F-TrCP-Ecad induction on the levels of the membrane and cytosolic β-catenin in the DLD1 colon cancer cells. β-catenin signaling is deregulated in DLD1 cells due to inactivation mutations of APC, resulting in the accumulation of unphosphorylated β-catenin resistant to βTrCP-mediated proteolysis. A stable DLD1-F-TrCP-Ecad cell line was constructed in which the engineered F-TrCP-Ecad was expressed under the tetracycline-repressible promoter [19]. The expression of F-TrCP-Ecad was induced when DLD1-F-TrCP-Ecad cells were cultured in the absence of doxycycline (Dox), a tetracycline analogue (Fig. 3C). To test whether induction of F-TrCP-Ecad affects the steady state levels of the membrane and cytosolic β-catenin, DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox, and fractionated into the cytosolic pool (S100) and the membrane pool (P100) by centrifugation. As seen in Fig. 3A, induction of F-TrCP-Ecad expression reduced the steady state level of cytosolic β-catenin, while it had no effect on the level of the membrane-associated β-catenin. In contrast, Dox treatment of the parental DLD1 cells had no effect on the steady state level of cytosolic β-catenin (data not shown). Therefore, F-TrCP-Ecad preferentially targets soluble nuclear/cytosolic β-catenin for degradation, sparing the membrane-associated fraction, which is tightly associated with endogenous membrane E-cadherin.


A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

F-TrCP-Ecad promotes β-catenin degradation and inhibits Wnt signaling in DLD1 cells. A. Effects of F-TrCP-Ecad on the levels of cytosolic and membrane-associated β-catenin. A DLD1 inducible cell line was generated with the tetracycline (tet)-off system; these cells expressed F-TrCP-Ecad only in the absence of Dox. Cells were grown in the presence or absence of Dox for 5 days, and subjected to subcellular fractionation. Equal amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the anti-β-catenin antibody. α-tubulin levels were determined by immunoblotting with the anti-α-tubulin antibody as an internal loading control. Quantified representation of immunoblot is shown at the bottom of each blot (relative intensity). B. F-TrCP-Ecad inhibits the transcriptional activity of β-catenin. DLD1-F-TrCP-Ecad cells were transfected with TOP-FLASH and a CMV-Renilla reporter, and cultured in the presence or absence of Dox. The luciferase activity of cells grown in the presence of Dox was arbitrarily set to 1. C. F-TrCP-Ecad induction inhibits c-MYC expression. DLD1-F-TrCP-Ecad cells were cultured in the presence or absence of Dox for four days. The expression level of c-myc and F-TrCP-Ecad were determined by immunoblotting with the anti-MYC (C-19) and the anti-FLAG antibodies (top and middle panels). Equal loadings were confirmed by immunoblotting with the anti-α-tubulin antibody (bottom panel). 'o' indicates two unknown species immuno-reactive with the anti-FLAG antibody, which migrated right below βTrCP-Ecad of the middle panel.
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Related In: Results  -  Collection

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Figure 3: F-TrCP-Ecad promotes β-catenin degradation and inhibits Wnt signaling in DLD1 cells. A. Effects of F-TrCP-Ecad on the levels of cytosolic and membrane-associated β-catenin. A DLD1 inducible cell line was generated with the tetracycline (tet)-off system; these cells expressed F-TrCP-Ecad only in the absence of Dox. Cells were grown in the presence or absence of Dox for 5 days, and subjected to subcellular fractionation. Equal amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the anti-β-catenin antibody. α-tubulin levels were determined by immunoblotting with the anti-α-tubulin antibody as an internal loading control. Quantified representation of immunoblot is shown at the bottom of each blot (relative intensity). B. F-TrCP-Ecad inhibits the transcriptional activity of β-catenin. DLD1-F-TrCP-Ecad cells were transfected with TOP-FLASH and a CMV-Renilla reporter, and cultured in the presence or absence of Dox. The luciferase activity of cells grown in the presence of Dox was arbitrarily set to 1. C. F-TrCP-Ecad induction inhibits c-MYC expression. DLD1-F-TrCP-Ecad cells were cultured in the presence or absence of Dox for four days. The expression level of c-myc and F-TrCP-Ecad were determined by immunoblotting with the anti-MYC (C-19) and the anti-FLAG antibodies (top and middle panels). Equal loadings were confirmed by immunoblotting with the anti-α-tubulin antibody (bottom panel). 'o' indicates two unknown species immuno-reactive with the anti-FLAG antibody, which migrated right below βTrCP-Ecad of the middle panel.
Mentions: We tested the effect of F-TrCP-Ecad induction on the levels of the membrane and cytosolic β-catenin in the DLD1 colon cancer cells. β-catenin signaling is deregulated in DLD1 cells due to inactivation mutations of APC, resulting in the accumulation of unphosphorylated β-catenin resistant to βTrCP-mediated proteolysis. A stable DLD1-F-TrCP-Ecad cell line was constructed in which the engineered F-TrCP-Ecad was expressed under the tetracycline-repressible promoter [19]. The expression of F-TrCP-Ecad was induced when DLD1-F-TrCP-Ecad cells were cultured in the absence of doxycycline (Dox), a tetracycline analogue (Fig. 3C). To test whether induction of F-TrCP-Ecad affects the steady state levels of the membrane and cytosolic β-catenin, DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox, and fractionated into the cytosolic pool (S100) and the membrane pool (P100) by centrifugation. As seen in Fig. 3A, induction of F-TrCP-Ecad expression reduced the steady state level of cytosolic β-catenin, while it had no effect on the level of the membrane-associated β-catenin. In contrast, Dox treatment of the parental DLD1 cells had no effect on the steady state level of cytosolic β-catenin (data not shown). Therefore, F-TrCP-Ecad preferentially targets soluble nuclear/cytosolic β-catenin for degradation, sparing the membrane-associated fraction, which is tightly associated with endogenous membrane E-cadherin.

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

Show MeSH
Related in: MedlinePlus