Ferroptosis, an iron and reactive oxygen species (ROS)-dependent non-apoptotic type of regulated cell death, is characterized by a massive iron overload and peroxidation of polyunsaturated fatty acids (PUFAs), which finally results in cell death. Recent studies suggest that ferroptosis can influence carcinogenesis negatively and therefore may be used as a novel anti-cancer strategy. Hepatocellular carcinoma (HCC) is a deadly malignancy with poor chances of survival and is the second leading cause of cancer deaths worldwide. Diagnosis at an already late stage and general resistance to current therapies may be responsible for the dismal outcome. As the liver acts as a key factor in iron metabolism, ferroptosis is shown to play an important role in HCC carcinogenesis and, more importantly, may hold the potential to eradicate HCC. In this review, we summarize the current knowledge we have of the role of ferroptosis in HCC and the application of ferroptosis as a therapy option and provide an overview of the potential translation of ferroptosis in the clinical practice of HCC.

Inhibition of histone deacetylases (HDACs) is a promising anti-cancer approach. For biliary tract cancer (BTC), only limited therapeutic options are currently available. Therefore, we performed a comprehensive investigation of HDAC expression and pharmacological HDAC inhibition into a panel of eight established BTC cell lines. The screening results indicate a heterogeneous expression of HDACs across the studied cell lines. We next tested the effect of six established HDAC inhibitors (HDACi) covering pan- and class-specific HDACis on cell viability of BTC cells and found that the effect (i) is dose- and cell-line-dependent, (ii) does not correlate with HDAC isoform expression, and (iii) is most pronounced for romidepsin (a class I HDACi), showing the highest reduction in cell viability with IC50 values in the low-nM range. Further analyses demonstrated that romidepsin induces apoptosis in BTC cells, reduces HDAC activity, and increases acetylation of histone 3 lysine 9 (H3K9Ac). Similar to BTC cell lines, HDAC 1/2 proteins were heterogeneously expressed in a cohort of resected BTC specimens (n = 78), and their expression increased with tumor grading. The survival of BTC patients with high HDAC-2-expressing tumors was significantly shorter. In conclusion, HDAC class I inhibition in BTC cells by romidepsin is highly effective in vitro and encourages further in vivo evaluation in BTC. In situ assessment of HDAC 2 expression in BTC specimens indicates its importance for oncogenesis and/or progression of BTC as well as for the prognosis of BTC patients.

Introduction Biliary tract cancer (BTC) is a fatal disease with limited therapeutic options. Inhibition of histone deacetylases (HDACs) represents an effective anti‐cancer approach. Data regarding HDAC and BTC are sparse but promising. There, we performed a comprehensive investigation of HDAC expression and pharmacological HDAC inhibition in a BTC in vitro model. Methods Expression of HDACs in BTC cells was measured via quantitative real‐time PCR and immunohistochemistry. Cytotoxicity of HDAC pan‐inhibitors (belinostat, vorinostat) and HDAC inhibitors targeting different HDAC classes (mocetinostat, romidepsin, LMK‐235, tubastatin A) was analyzed using the resazurin assay. HDAC1/2 activity was measured via the HDAC‐Glo I/II Assay and Screening System (Promega). Mode of cytotoxicity of romidepsin was evaluated via a time‐resolved resazurin assay and the RealTime‐Glo Annexin V Apoptosis and Necrosis Assay (Promega). Changes in H3K9Ac levels were measured to investigate the epigenetic effect of romidepsin. Combinatory treatment of romidepsin and cisplatin was evaluated via the resazurin assay and changes of IC50‐values. Expression of HDAC1/2 in n = 78 BTC patient samples was done via immunohistochemistry. Appropriate statistical tests were used to correlate HDAC1/2 expression with clinicopathological data. Results HDAC profiling revealed a heterogeneous expression of HDACs across the studied cell lines (n = 8). Furthermore, we found that BTC cells show different sensitivities towards the used HDAC inhibitors. The HDAC class I inhibitor romidepsin showed significant cytotoxicity in the (low) nM‐range and was therefore chosen for further investigation. We found that romidepsin causes apoptosis and secondary necrosis and significantly reduces HDAC activity in BTC cells. Furthermore, we observed that sub‐lethal concentrations of romidepsin augmented the toxic effect of the standard chemotherapeutic cisplatin. We also measured HDAC1 and 2 expression in BTC patients. HDAC1 expression correlated with tumor localization and tumor grading. Moreover, patients with high expression of HDAC2 had a significant shorter overall survival time. Conclusions Our results demonstrate that the HDAC class I inhibitor romidepsin is a promising anti‐BTC substance. In addition, we show that HDAC1 and 2 are expressed in BTC patient samples and associate with clinical parameters.

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.

The term long non-coding RNA (lncRNA) describes non protein-coding transcripts with a length greater than 200 base pairs. The ongoing discovery, characterization and functional categorization of lncRNAs has led to a better understanding of the involvement of lncRNAs in diverse biological and pathological processes including cancer. Aberrant expression of specific lncRNA species was demonstrated in various cancer types and associated with unfavorable clinical characteristics. Recent studies suggest that lncRNAs are also involved in the development and progression of biliary tract cancer, a rare disease with high mortality and limited therapeutic options. In this review, we summarize current findings regarding the manifold roles of lncRNAs in biliary tract cancer and give an overview of the clinical and molecular consequences of aberrant lncRNA expression as well as of underlying regulatory functions of selected lncRNA species in the context of biliary tract cancer.

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.

Introduction:Although molecular insights about biliary tract cancer (BTC) increased in the last decade, new therapeutic strategy like inhibition of histone deacetylases (HDACs) could additionally improve the still dismal outcome of this tumor entity. Methods: Therefore, we performed comprehensive investigation of HDAC expression and pharmacological inhibition in a panel of eight established BTC cell lines and in a cohort resected native BTC specimens (n = 78). Results: HDAC profiling revealed a heterogeneous expression of HDACs across the studied cell lines and the BTC cancer specimen. Cytotoxicity of six established HDAC inhibitors (HDACi) covering pan-and class-specific HDACis was dose-as well as cell line-dependent and did not show a statistical correlation with HDAC isoform expression. Romidepsin (a class II HDACi), induced the highest reduction of cell viability and apoptosis in BTC cells which was paralleled by reducing HDAC1/2 activity and increasing histone 3 lysine 9 acetylation. Furthermore, non-toxic concentrations of romidepsin could augment the cytotoxic effect of the standard chemotherapeutic cisplatin. Related to the clinical tumor specimen, HDAC expression pattern correlated with the tumor grading and the survival of BTC patients. Conclusions: In conclusion, in-vitro-experiments provide clear evidence that the HDAC class I inhibitor romidepsin is effective for BTC alone and acts supportively in combination with standard chemotherapeutics. Additionally, the observed HDAC expression in BTC specimens could serve as a predictive and prognostic biomarker for BTC patients.