Biliary tract cancer (BTC) is a gastrointestinal malignancy associated with a poor survival rate. Current therapies encompass palliative and chemotherapeutic treatment as well as radiation therapy, which results in a median survival of only one year due to standard therapeutic ineffectiveness or resistance. Tazemetostat is an FDA-approved inhibitor of enhancer of Zeste homolog 2 (EZH2), a methyltransferase involved in BTC tumorigenesis via trimethylation of histone 3 at lysine 27 (H3K27me3), an epigenetic mark associated with silencing of tumor suppressor genes. Up to now, there are no data available regarding tazemetostat as a possible treatment option against BTC. Therefore, the aim of our study is a first-time investigation of tazemetostat as a potential anti-BTC substance in vitro. In this study, we demonstrate that tazemetostat affects cell viability and the clonogenic growth of BTC cells in a cell line-dependent manner. Furthermore, we found a strong epigenetic effect at low concentrations of tazemetostat, which was independent of the cytotoxic effect. We also observed in one BTC cell line that tazemetostat increases the mRNA levels and protein expression of the tumor suppressor gene Fructose-1,6-bisphosphatase 1 (FBP1). Interestingly, the observed cytotoxic and epigenetic effects were independent of the mutation status of EZH2. To conclude, our study shows that tazemetostat is a potential anti-tumorigenic substance in BTC with a strong epigenetic effect.

Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.

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