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

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 4

EBP50 interacts with TCF-1 and β-catenin through its first and second PDZ domains, respectively.

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EBP50 interacts with TCF-1 and β-catenin through its first and second PD...
(A) Cell lysates from HEK293 cells overexpressing Myc–TCF-1 or HA–TCF-4 were incubated with glutathione beads loaded with GST, GST-EBP50 (NHERF1), or GST-NHERF2. After washing, bound materials were analyzed by immunoblotting. (B) In vitro GST pull-down assays employing purified His-EBP50 and GST, GST–dnTCF-1, or GST–dnTCF-1dc, a PDZ motif deletion mutant. (C) GST-fused full-length EBP50 (GST-EBP50) and the first (PDZ1) and second PDZ domains (PDZ2) of EBP50 were mixed with equal amounts of lysates from MDCK cells stably expressing Myc–dnTCF-1. After washing, bound materials were examined by immunoblotting. Lower panels of A–C: Coomassie blue–stained gels demonstrated the amount of each purified GST fusion protein used in the assays. (D) NCI-H28 lysates were immunoprecipitated by either control or anti-EBP50 antibody, followed by immunoblotting using TCF-1 or EBP50 antibodies. (E) Cellular lysates from Colo205 cells, which expressed both full-length (*) and N-terminally truncated TCF-1 (**), were incubated with GST or GST-EBP50 beads, and the bound material was analyzed by immunoblotting using anti–TCF-1 antibody. (F) Colo205 cell lysates were incubated with mouse anti-EBP50 or control antibody conjugated on NHS-activated beads and then processed for immunoblotting using rabbit anti-EBP50 and TCF-1 antibodies. (G) Top row: Double immunofluorescence and confocal microscopy revealed the localization of endogenous β-catenin (green) and EBP50 (red) in SW480 cells. Bottom: SW480 cells were transfected with Myc–TCF-1. Anti-Myc staining showed the colocalization of TCF-1 with endogenous EBP50 in the nucleus. Scale bars: 10 μm.