Abstract Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines (Exp Cell Res 2011). The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012). However, K cells often showed morphological alteration, reminiscent of epithelial-mesenchymal transition (EMT), which is involved in non-canonical Wnt signaling (BMC Cancer 2013). The finding suggested that the SxxSS motif had potential to regulate EMT through the non-canonical Wnt signaling pathway. Thus, the AIM of this study was to investigate whether the SxxSS motif modulated expression levels of EMT regulators and Wnt5a, a representative non-canonical Wnt ligand. Methods: The human HCC cell line HAK-1A (Hepatology 1993) was used. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Sh-RNA was used to knockdown wnt5a gene expression. Results: The 269A-mutant cells robustly expressed Wnt5a in both protein and mRNA levels, while empty vector-transfected cells (control), J cells, or K cells did not. Of note, Wnt5a expression was coupled with SLUG expression and EMT-like cellular morphological change. Snail was hardly expressed in the cells examined in this study. When the Wnt5a expression was specifically silenced by using sh-RNA, the expression level of SLUG was clearly decreased. Conclusion: The findings in this study suggest that serine 269 (the first serine) of the SxxSS motif of TCF-4 is a major switch to control Wnt5a transcription, thereby modulating the expression level of the EMT-regulator SLUG in a human HCC cell line. Citation Format: Hironori Koga, Toru Nakamura, Mitsuhiko Abe, Yu Ikezono, Fumitaka Wada, Hirohisa Yano, Takuji Torimura. The SxxSS motif of T-cell Factor-4 isoforms regulates Wnt5a expression and EMT in human liver cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2015-1436