Claudin-2 extracellular loop 1-mimetic peptides functionally inhibit combined hepatocellular-cholangiocarcinoma cells

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare and aggressive liver cancer for which effective targeted therapies have not been established. We analyzed claudin expression in cHCC-CCA cell lines (KMCH-1 and KMCH-2) and found that claudin-2 was markedly overexpressed relative to hepatocellular carcinoma and cholangiocarcinoma cell lines, as well as primary hepatocytes. This upregulation was associated with the Wnt/β-catenin signaling pathway, a major regulator of cell proliferation. Claudin-2 knockdown reduced cell viability in both cHCC-CCA cell lines examined, suggesting that claudin-2 supports viability in these cell line models. To test whether claudin-2 function can be modulated extracellularly, we synthesized peptides mimicking the first extracellular loop (ECL1) of claudin-2. Among them, the C-terminal peptide Ex1C selectively reduced the viability of cHCC-CCA cell lines examined, but not that of other cells lacking claudin-2 overexpression. Ex1C suppressed proliferation without inducing cell death and significantly inhibited adhesion of KMCH-2 and KMCH-1 cells to primary hepatocytes. As Ex1C exhibited homotypic self-association, these effects are consistent with interference with ECL1-dependent claudin-2 interactions. Collectively, our findings identify claudin-2 as a characteristic molecular feature of the examined cHCC-CCA cell lines and support the utility of extracellular loop-mimetic peptides as probes to dissect claudin-dependent regulation of proliferation and cell adhesion.