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

BioMed CentralBMC Molecular Biology ssOpen AcceResearch article A protein knockdown strategy to study the function of β-catenin in tumorigenesis Feng Cong*1, Jianxuan Zhang2, William Pao1, Pengbo Zhou*2 and Harold Varmus1 Address: 1Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA and 2Department of Pathology, Weill Medical College and Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA Email: Feng Cong* - congf@; Jianxuan Zhang - jiz2001@; William Pao - paow@; Pengbo Zhou* - pez2001@; Harold Varmus - varmus@ * Corresponding authors 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 wer