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Aberrant nuclear localization of EBP50 promotes colorectal carcinogenesis in xenotransplanted mice by modulating TCF-1 and β-catenin interactions
Yu-Yu Lin, … , Chi-Ling Chen, Tzuu-Shuh Jou
Yu-Yu Lin, … , Chi-Ling Chen, Tzuu-Shuh Jou
Published April 2, 2012
Citation Information: J Clin Invest. 2012;122(5):1881-1894. https://doi.org/10.1172/JCI45661.
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Research Article Oncology Article has an altmetric score of 1

Aberrant nuclear localization of EBP50 promotes colorectal carcinogenesis in xenotransplanted mice by modulating TCF-1 and β-catenin interactions

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Abstract

Dysregulation of canonical Wnt signaling is thought to play a role in colon carcinogenesis. β-Catenin, a key mediator of the pathway, is stabilized upon Wnt activation and accumulates in the nucleus, where it can interact with the transcription factor T cell factor (TCF) to transactivate gene expression. Normal colonic epithelia express a truncated TCF-1 form, called dnTCF-1, that lacks the critical β-catenin–binding domain and behaves as a transcriptional suppressor. How the cell maintains a balance between the two forms of TCF-1 is unclear. Here, we show that ERM-binding phosphoprotein 50 (EBP50) modulates the interaction between β-catenin and TCF-1. We observed EBP50 localization to the nucleus of human colorectal carcinoma cell lines at low cell culture densities and human primary colorectal tumors that manifested a poor clinical outcome. In contrast, EBP50 was primarily membranous in confluent cell lines. Aberrantly located EBP50 stabilized conventional β-catenin/TCF-1 complexes and connected β-catenin to dnTCF-1 to form a ternary molecular complex that enhanced Wnt/β-catenin signaling events, including the transcription of downstream oncogenes such as c-Myc and cyclin D1. Genome-wide analysis of the EBP50 occupancy pattern revealed consensus binding motifs bearing similarity to Wnt-responsive element. Conventional chromatin immunoprecipitation assays confirmed that EBP50 bound to genomic regions highly enriched with TCF/LEF binding motifs. Knockdown of EBP50 in human colorectal carcinoma cell lines compromised cell cycle progression, anchorage-independent growth, and tumorigenesis in nude mice. We therefore suggest that nuclear EBP50 facilitates colon tumorigenesis by modulating the interaction between β-catenin and TCF-1.

Authors

Yu-Yu Lin, Yung-Ho Hsu, Hsin-Yi Huang, Yih-Jyh Shann, Chi-Ying F. Huang, Shu-Chen Wei, Chi-Ling Chen, Tzuu-Shuh Jou

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Figure 2

Nuclear EBP50 modulates Wnt/β-catenin signaling.

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Nuclear EBP50 modulates Wnt/β-catenin signaling.
(A) SW480 cells were in...
(A) SW480 cells were independently transfected with 40 μM siE50-1 or siE50-2, two siRNAs targeted at discrete regions of EBP50 cDNA, for 16 hours. Then, cells were trypsinized and replated into 12-well plates for 8 hours before being transfected with Top/Fop-flash reporter plasmids plus 40 μM of each EBP50 siRNA. After an additional 24 hours, cells were processed for luciferase activity assays. Western blotting demonstrated that both siRNAs efficiently suppressed the expression of EBP50. (B) The molecular configuration of the plasmids used in C and D. (C) HEK293 cells were transfected with the indicated plasmids, using empty vector pcDNA3 as a control (Ctr), and 1.5 μg of Top/Fop-flash reporter plasmid. Luciferase activity was assessed 24 hours later in triplicate. The data shown in A and C are mean ± SEM of 3 independent assays, each performed in triplicate. *P < 0.05, **P < 0.001. (D) EBP50-knockdown SW480 cells were transfected with the indicated FLAG-tagged EBP50 plasmids for 6 hours and stained with rabbit anti-FLAG (green) and mouse anti–β-catenin (red) antibodies. Scale bars: 10 μm.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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