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. The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. Furthermore, we have shown that TCF-4J predominantly expressed in poorly differentiated HCC tissues and conferred high tumorigenic potential to HCC cells (Koga et al., PLoS ONE 2012). In contrast, TCF-4K showed low tumorigenic potential, which is an important feature of cancer stem cells (CSCs). Another characteristic of CSC is robust resistance to anticancer agents. Among the agents, doxorubicin (DXR) has been a key drug applied to HCC treatment through a unique nanoparticle-mediated drug delivery system. Thus, we aimed to assess if TCF-4J and K differentially contributed to resistance to DXR-induced HCC cell apoptosis, focusing on the expression profile of Bcl-xL, since the β-catenin/TCF-4 complex is known to up-regulate Bcl-xL gene expression.

Methods: The human HCC cell line HAK-1A 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. Stable cell clones overexpressing TCF-4J (J cells), K (K cells), and K-mutants (269A, 272A, and 273A cells, respectively) were established. The cells were incubated with DXR at 0, 100, 500, and 1000 ng/mL over 4 days. A MTS assay and Western blot analysis were performed to evaluate cell proliferation and the apoptosis-related protein levels of cleaved PARP, cleaved caspase-3, and Bcl-xL, respectively. Flow cytometric analysis was employed to determine the sub-G1 population.

Results: Dose-dependent apoptotic effects were verified by PARP cleavage, caspase activation, and increase in the sub-G1 population. Unexpectedly, K cells, harboring the SxxSS motif, were most resistant to DXR. Under 1000 ng/mL of DXR treatment, the absorbance ratio of viable cells at Day 3 to that at Day 0 in control cells, J cells, and K cells were 1.15, 1.53, and 1.76, respectively. However, the K-mutant 269A cells lost high resistance, and the ratio fell to 1.11. Consistent with the results, the expression level of Bcl-xL was highest in K cells. The Bcl-xL protein level of 269A mutant cells was found similar to that of mock DNA transfected cells.

Conclusions: The findings suggest that the SxxSS motif of TCF-4 is involved in resistance to DXR, by modulating the expression level of Bcl-xL. We suggest that phosphorylation at serine 269 in the TCF-4K isoform is critical to promote anti-apoptotic potential through increasing the Bcl-xL expression in HCC.

Citation Format: Hironori Koga, Miran Kim, Yoshito Tomimaru, Toru Nakamura, Mitsuhiko Abe, Osamu Hashimoto, Yu Ikezono, Hiroshi Masuda, Hirohisa Yano, Takato Ueno, Takuji Torimura, Jack R. Wands, Michio Sata. Human TCF-4 isoforms regulate apoptosis through upregulating Bcl-xL expression in a SxxSS motif-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 890. doi:10.1158/1538-7445.AM2013-890