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

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 3

EBP50 binds to the TCF/LEF consensus motif.

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EBP50 binds to the TCF/LEF consensus motif. (A) Mock- and an EBP50 siRNA...
(A) Mock- and an EBP50 siRNA–transfected SW480 clone (siE50#20) were processed for ChIP assay using anti-EBP50 antibody. The ligation-mediated PCR (LM-PCR) products from genomic (input) and anti-EBP50 antibody immunoprecipitated (ChIP) DNA are shown. (B) Ten motifs with the highest scores, compiled from the 57 most significantly EBP50-bound regions, as predicted by a BioProspector Search, are shown. Among them, 6 motifs identical or similar to the TCF/LEF consensus motif are shown in bold. (C) Molecular structure of TCF family proteins. HMG, high-mobility group proteins; NLS, nucleus localization signal. The last 4 amino acids at the C-terminal ends are shown in bold if those sequences match to a potential PDZ-binding motif. (D) SW480 cells were transfected with 2 μg of the indicated plasmids, using empty vector as a control, and 0.4 μg of Top/Fop-flash reporter plasmids. TCF-1dc is a C-terminally deleted TCF-1 construct lacking the EBP50 binding motif. Luciferase activity was assessed and presented as in Figure 2C. *P < 0.05. (E) SW480 cells were processed for ChIP assay using the indicated antibodies or a rabbit anti-podocalyxin antibody as a control. Selective Wnt/β-catenin–regulated gene sequences were amplified by PCR. *Genes reproducibly identified as Wnt/β-catenin targets by two previous systemic ChIP analyses (39, 40) as well as EBP50 binding sites by our ChIP-on-chip assay. **This gene, although not demonstrated to be Wnt/β-catenin regulated with vigorous experimental evidence, was revealed by previous systemic analysis (39, 40) and this study.