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

Authors: Cong, F.; Zhang, J.; Pao, W.; Zhou, P.; Varmus, H.
Article Title: A protein knockdown strategy to study the function of β-catenin in tumorigenesis
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 (Skp1, Cullin 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. ©2003 Cong et al; licensee BioMed Central Ltd.
Keywords: signal transduction; controlled study; protein expression; gene mutation; human cell; proto-oncogene proteins; nonhuman; protein function; protein localization; cell proliferation; mouse; animals; mice; gene targeting; cell division; cell growth; ubiquitin protein ligase; protein degradation; protein metabolism; transcription initiation; animal experiment; animal model; protein binding; cell fate; in vitro study; cell line, tumor; uvomorulin; carcinogenesis; mus musculus; colorectal neoplasms; ubiquitination; recombinant fusion proteins; mice, nude; colon cancer; myc protein; tumor cell; cellular distribution; trans-activators; oncogene c myc; wnt proteins; beta catenin; wnt protein; ubiquitins; cyclin d; cytoskeletal proteins; chimeric protein; colon polyposis; zebrafish proteins; protein engineering; human; article
Journal Title: BMC Molecular Biology
Volume: 4
ISSN: 1471-2199
Publisher: Biomed Central Ltd  
Date Published: 2003-09-29
Start Page: Article 10
Language: English
DOI: 10.1186/1471-2199-4-10
PUBMED: 14516475
PROVIDER: scopus
PMCID: PMC222962
Notes: Export Date: 12 September 2014 -- Source: Scopus
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MSK Authors
  1. William Pao
    138 Pao
  2. Feng Cong
    6 Cong
  3. Harold Varmus
    94 Varmus