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Abstract 4029: The Wnt target geneCLAUDIN-2regulates tumorigenesis and stemness in human liver cancer cells

Background: In our previous studies on T-cell factor (TCF)-4 isoforms, we identified a unique target gene CLAUDIN-2, which was upregulated by lack of SxxSS motif in exon 9 of TCF-4 (Exp Cell Res 2011, Liver Int 2013). This type of TCF-4 (TCF-4J) conferred high tumorigenic ability and hypoxia resistance to hepatocellular carcinoma (HCC) cells (PLoS One 2012). Of interest, the TCF-4J isoform-overexpressing HCC cells (J cells) strongly expressed the leaky tight junction protein CLAUDIN-2 under sphere-forming conditions, suggesting that J cells have cancer stem cell (CSC)-like features involving CLAUDIN-2 upregulation. Thus, the Aim of this study was to assess expression profile of CLAUDIN-2 and the CSC-relevant function of the molecule, such as tumorigenicity, in liver cancer cells. Methods: The human liver cancer cell lines used in this study were as follows: HAK-1A, HAK-1B (Hepatology 1993), Huh-7, HepG2, Hep3B, HLF, KMCH-1 (Hepatology 1987), KMCH-2 (J Hepatol 1996), KYN-1, KYN-2, and HAK-5. KMCH-1 and -2 are unique combined hepatocellular-cholangiocarcinoma cell lines, and HAK-5 is a sarcomatous HCC cell line. Lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) and plasmid-mediated CLAUDIN-2 cDNA (Origene) were utilized to generate stable knock-down (KD) and overexpressing cells, respectively. Results: CLAUDIN-2 was exclusively expressed in both KMCH-1 and KMCH-2 cells, showing 240~370-fold mRNA level of that in the well-differentiated HCC line HAK-1A. The protein expression was strongly observed at cytoplasmic membrane and cytoplasm in KMCH-1 and KMCH-2 cells, but not in the others examined. Gene silencing of CLAUDIN-2 resulted in slower cell proliferation. In xenograft experiment in nude mice, CLAUDIN-2-KD KMCH-2 cells formed much smaller tumors in size than the control cells. Of interest, overexpression of CLAUDIN-2 in constitutively low expressing cell lines, such as Hep3B and HLF cells, lead to high expression of Nanog, a stemness gene product. Conclusion: As previously shown in transformed HCC cells by the Wnt transcription factor TCF-4J, CLAUDIN-2 was a key molecule regulating cell proliferation and tumorigenesis in non-gene-engineered human combined hepatocellular-cholangiocarcinoma cells. To elucidate precise mechanism of exclusively high expression of this molecule in such difficult-to-treat type of liver cancer may provide insights into generating novel therapeutic approaches. Citation Format: Hironori Koga, Fumitaka Wada, Mitsuhiko Abe, Hideki Iwamoto, Toru Nakamura, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Hirohisa Yano, Takuji Torimura. The Wnt target gene CLAUDIN-2 regulates tumorigenesis and stemness in human liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4029.