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Open Access Highly Accessed Research article

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

Feng Cong1*, Jianxuan Zhang2, William Pao1, Pengbo Zhou2* and Harold Varmus1

Author Affiliations

1 Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

2 Department of Pathology, Weill Medical College and Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA

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BMC Molecular Biology 2003, 4:10  doi:10.1186/1471-2199-4-10

Published: 29 September 2003

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 β-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 β-catenin or adenomatous polyposis coli (APC), renders β-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 β-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the β-catenin binding domain of E-cadherin fused to βTrCP ubiquitin-protein ligase, the stable β-catenin mutant was recruited to the cellular SCF (

    S
kp1,
    C
ullin 1, and
    F
-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type β-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of βTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of β-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 β-catenin. Our results suggest that a high concentration of cytoplasmic β-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.