Background: We previously identified a unique Wnt target gene CLAUDIN-2, whose expression level was regulated by isoforms of the Wnt signal transcription factor T-cell factor-4 (Exp Cell Res 2011, Liver Int 2013). CLAUDIN-2 (CLDN-2) was highly expressed in combined hepatocellular-cholangiocarcinoma cell lines, and the molecule contributed to high cellular proliferation, sphere-forming ability, and tumorigenicity of the cells (Koga et al., The Liver Meeting 2018). However, such tumor-promoting action of CLDN-2 was not demonstrated in all liver cancer cell lines tested. The opposite functions of CLDN-2 included extremely low proliferative activity and no tumorigenicity, that were, at least, associated with the tumor suppressor LKB1-mediated AMPK activation.

Thus, the Aim of this study was to further investigate molecular basis for the two-faced actions of CLDN-2 in liver cancer cells.

Methods: The human liver cancer cell lines Hep3B and HLF were used in this study. Plasmid-mediated CLAUDIN-2 cDNA (Origene) and lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) were utilized to generate stable overexpressing and knock-down (KD) cells, respectively. A cDNA microarray analysis and qPCR were employed for exploring genetic determinants that controlled the two-faced actions of CLDN-2.

Results: CLDN-2-overexpressing HLF (CLDN-2-OE-HLF) cells exhibited clear G1 cell-cycle arrest and no tumorigenicity, although CLDN-2-OE-Hep3B cells did not. The cDNA microarray analysis comparing CLDN-2-OE-HLF cells with CLDN-2-OE-Hep3B cells and the validation qPCR revealed 7.9-fold increase of LRP4 and 1.6-fold increase of APC in the CLDN-2-OE-HLF cells, while 0.4-fold and 0.7-fold in the latter, respectively. Of interest, in western blot analysis, the CLDN-2-OE-HLF cells exhibited increased expression of the stem cell marker Nanog, in concert with those of phosphorylated (p-)LKB1 and p-AMPKalpha1, that together resulted in upregulation of p53 and p21 in both mRNA and protein levels.

Conclusion: LRP4 has been reported to have suppressive function on the Wnt signaling pathway interplaying with Frizzled at cell membrane, and APC is a strong tumor suppressor to inhibit beta-catenin-mediated Wnt signaling. Therefore, the findings obtained from this study suggested that the Wnt target gene CLDN-2 differentially regulated feedforward and feedback signal relays in the Wnt signal pathway in a context-dependent manner in human liver cancer cells.

Citation Format: Hironori Koga, Yasuko Imamura, Toru Nakamura, Hideki Iwamoto, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Dan Nakano, Hiroyuki Suzuki, Hirohisa Yano, Takuji Torimura. Opposite functions of Claudin-2 involving Wnt signaling in liver cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2627.