Aberrant nuclear localization of EBP50 promotes colorectal carcinogenesis in xenotransplanted mice by modulating TCF-1 and β-catenin interactions
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
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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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 5

EBP50 promotes interaction of β-catenin and dnTCF-1.

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EBP50 promotes interaction of β-catenin and dnTCF-1. (A) SW480 cells wer...
(A) SW480 cells were transfected with 2 μg of the indicated plasmids and 0.4 μg of Top/Fop-flash reporter plasmids before luciferase activity was assessed as in Figure 2C. *P < 0.05. (B) SW480 cells stably expressing FLAG–dnTCF-1 were immunoprecipitated by either control or anti-FLAG antibody, followed by FLAG peptide elution. The eluate was then processed for Re-ChIP assay using EBP50 and β-catenin antibodies, respectively, and using rabbit anti-podocalyxin antibody as a control. Input represented 10% of total chromatin used for each experiment. (C) Purified Trx-His–fused full-length EBP50 (E50) and the first (PDZ1) and second PDZ domains (PDZ2) of EBP50 were incubated with glutathione beads conjugated with GST–dnTCF-1 (top panel) and GST–β-catenin C-terminal (middle panel) recombinant proteins. After incubation and washing, bound materials were assessed by immunoblotting. The amount and purity of the recombinant proteins are shown by a Coomassie blue–stained gel in the bottom panel. (D) Glutathione beads conjugated with GST–dnTCF-1 were incubated with the indicated Trx-His fusion proteins for 1 hour. Then untagged β-catenin, which was released from the GST–β-catenin by thrombin digestion, was added in the mixture for 1 more hour before the pulled-down materials were examined by immunoblotting. (E) SW480 cells stably expressing FLAG–dnTCF-1 were infected with lentivirus expressing siRNA against EBP50 (shE50) or luciferase control (shLuc). The cellular lysates were immunoprecipitated by mouse anti-FLAG antibody and processed for immunoblotting using the indicated antibodies. (F) Colo205 cells were infected with lentivirus expressing siRNA against EBP50 or luciferase control. The cellular lysates were immunoprecipitated by rabbit anti–β-catenin antibody and processed for immunoblotting using mouse anti–β-catenin, anti-EBP50, and anti–TCF-1 antibodies.