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 7

Tumorigenesis of the colon cancer cells is reduced by EBP50 knockdown, which disrupted β-catenin and TCF-1 interaction.

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Tumorigenesis of the colon cancer cells is reduced by EBP50 knockdown, w...
(A) Mock and EBP50 siRNA stably transfected (siE50) SW480 clones were injected beneath the dorsal skin of 6-week-old NOD-SCID mice. After 3 weeks, the mice were sacrificed. The injected tumors (n = 5 for each group) were dissected from the mice and weighed, and the results are shown (mean ± SEM). (B) Mock and EBP50 siRNA stably transfected HT29 clones were processed for soft agar assay to assess their anchorage-independent growth ability. The histogram shows results (mean ± SEM) from 3 different fields for each clone. The inset shows the result from Western blot analysis of EBP50 in mock and siRNA-expressing clones. *P < 0.05, **P < 0.001. (C) Cell lysates from mock and siE50 SW480 clone were immunoprecipitated with equal amounts of mouse normal serum (Ctr) and anti–β-catenin monoclonal antibody and processed for Western blot analysis using the indicated antibodies. (D) Mock (M) or siEBP50 SW480 (Si) cells were processed for ChIP assay using rabbit anti-EBP50, anti–β-catenin, or anti–TCF-1 antibody. Five percent of the immunoprecipitated DNA served as the templates for amplifying all the target sequences by PCRs, except those for MMP2, which used 0.5% of the immunoprecipitated materials as the templates for PCRs.