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


CONCLUSIONS
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.
D000818:Animals; D002455:Cell Division; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008819:Mice, Nude; D015202:Protein Engineering / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014452:Ubiquitins / Q000378:metabolism; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin
C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014452:Ubiquitins; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin