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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 inhibits the transcriptional activity of β-catenin S37A. A. 293 cells were co-transfected with a β-catenin S37A expression plasmid, indicated βTrCP expression plasmids, TOP-FLASH, and a CMV-Renilla reporter. The luciferase activities were measured and normalized against the control Renilla activities (top panel). The luciferase activity of cells transfected with βTrCP was arbitrarily set to 1. The expression of βTrCP derivatives was determined by immunoblotting with the anti-FLAG antibody (bottom panel). B. F-TrCP-Ecad and F-TrCP(ΔN)-Ecad bind to β-catenin to the similar extent. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by SDS-PAGE and immunoblotted with the anti-HA antibody (top panel). The expression of FLAG-tagged βTrCP derivatives and β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-FLAG and anti-HA antibodies (middle and bottom panels).
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Figure 2: F-TrCP-Ecad inhibits the transcriptional activity of β-catenin S37A. A. 293 cells were co-transfected with a β-catenin S37A expression plasmid, indicated βTrCP expression plasmids, TOP-FLASH, and a CMV-Renilla reporter. The luciferase activities were measured and normalized against the control Renilla activities (top panel). The luciferase activity of cells transfected with βTrCP was arbitrarily set to 1. The expression of βTrCP derivatives was determined by immunoblotting with the anti-FLAG antibody (bottom panel). B. F-TrCP-Ecad and F-TrCP(ΔN)-Ecad bind to β-catenin to the similar extent. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by SDS-PAGE and immunoblotted with the anti-HA antibody (top panel). The expression of FLAG-tagged βTrCP derivatives and β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-FLAG and anti-HA antibodies (middle and bottom panels).

Mentions: We further tested the effect of F-TrCP-Ecad on the transcriptional activity of β-catenin. β-catenin S37A was transiently expressed in 293 cells with TOP-FLASH, a luciferase reporter that contains multiple copies of TCF binding sites and serves as a readout for β-catenin signaling activity [18]. As shown in Fig. 2A, co-expression of F-TrCP-Ecad dramatically decreased the transcriptional response to β-catenin S37A from the TOP-FLASH reporter. Two other β-catenin targeting peptides, which constitute the N-terminal domains of human TCF4 or Xenopus TCF3, were also fused to βTrCP, and demonstrated similar inhibitory effects on β-catenin signaling as F-TrCP-Ecad (data not shown).


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 inhibits the transcriptional activity of β-catenin S37A. A. 293 cells were co-transfected with a β-catenin S37A expression plasmid, indicated βTrCP expression plasmids, TOP-FLASH, and a CMV-Renilla reporter. The luciferase activities were measured and normalized against the control Renilla activities (top panel). The luciferase activity of cells transfected with βTrCP was arbitrarily set to 1. The expression of βTrCP derivatives was determined by immunoblotting with the anti-FLAG antibody (bottom panel). B. F-TrCP-Ecad and F-TrCP(ΔN)-Ecad bind to β-catenin to the similar extent. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by SDS-PAGE and immunoblotted with the anti-HA antibody (top panel). The expression of FLAG-tagged βTrCP derivatives and β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-FLAG and anti-HA antibodies (middle and bottom panels).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC222962&req=5

Figure 2: F-TrCP-Ecad inhibits the transcriptional activity of β-catenin S37A. A. 293 cells were co-transfected with a β-catenin S37A expression plasmid, indicated βTrCP expression plasmids, TOP-FLASH, and a CMV-Renilla reporter. The luciferase activities were measured and normalized against the control Renilla activities (top panel). The luciferase activity of cells transfected with βTrCP was arbitrarily set to 1. The expression of βTrCP derivatives was determined by immunoblotting with the anti-FLAG antibody (bottom panel). B. F-TrCP-Ecad and F-TrCP(ΔN)-Ecad bind to β-catenin to the similar extent. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by SDS-PAGE and immunoblotted with the anti-HA antibody (top panel). The expression of FLAG-tagged βTrCP derivatives and β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-FLAG and anti-HA antibodies (middle and bottom panels).
Mentions: We further tested the effect of F-TrCP-Ecad on the transcriptional activity of β-catenin. β-catenin S37A was transiently expressed in 293 cells with TOP-FLASH, a luciferase reporter that contains multiple copies of TCF binding sites and serves as a readout for β-catenin signaling activity [18]. As shown in Fig. 2A, co-expression of F-TrCP-Ecad dramatically decreased the transcriptional response to β-catenin S37A from the TOP-FLASH reporter. Two other β-catenin targeting peptides, which constitute the N-terminal domains of human TCF4 or Xenopus TCF3, were also fused to βTrCP, and demonstrated similar inhibitory effects on β-catenin signaling as F-TrCP-Ecad (data not shown).

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