TY - JOUR A1 - Sarma, Bhavishya A1 - Willmes, Christoph A1 - Angerer, Laura A1 - Adam, Christian A1 - Becker, Jürgen C. A1 - Kervarrec, Thibault A1 - Schrama, David A1 - Houben, Roland T1 - Artesunate affects T antigen expression and survival of virus-positive Merkel cell carcinoma JF - Cancers N2 - Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer with frequent viral etiology. Indeed, in about 80% of cases, there is an association with Merkel cell polyomavirus (MCPyV); the expression of viral T antigens is crucial for growth of virus-positive tumor cells. Since artesunate — a drug used to treat malaria — has been reported to possess additional anti-tumor as well as anti-viral activity, we sought to evaluate pre-clinically the effect of artesunate on MCC. We found that artesunate repressed growth and survival of MCPyV-positive MCC cells in vitro. This effect was accompanied by reduced large T antigen (LT) expression. Notably, however, it was even more efficient than shRNA-mediated downregulation of LT expression. Interestingly, in one MCC cell line (WaGa), T antigen knockdown rendered cells less sensitive to artesunate, while for two other MCC cell lines, we could not substantiate such a relation. Mechanistically, artesunate predominantly induces ferroptosis in MCPyV-positive MCC cells since known ferroptosis-inhibitors like DFO, BAF-A1, Fer-1 and β-mercaptoethanol reduced artesunate-induced death. Finally, application of artesunate in xenotransplanted mice demonstrated that growth of established MCC tumors can be significantly suppressed in vivo. In conclusion, our results revealed a highly anti-proliferative effect of the approved and generally well-tolerated anti-malaria compound artesunate on MCPyV-positive MCC cells, suggesting its potential usage for MCC therapy. KW - artesunate KW - Merkel cell carcinoma KW - MCC KW - polyomavirus KW - ferroptosis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203851 SN - 2072-6694 VL - 12 IS - 4 ER - TY - JOUR A1 - Orouji, Elias A1 - Peitsch, Wiebke K. A1 - Orouji, Azadeh A1 - Houben, Roland A1 - Utikal, Jochen T1 - Unique role of histone methyltransferase PRDM8 in the tumorigenesis of virus-negative Merkel cell carcinoma JF - Cancers N2 - Merkel cell carcinoma (MCC) is a deadly skin cancer, and about 80% of its cases have been shown to harbor integrated Merkel polyomavirus in the tumor cell genome. Viral oncoproteins expressed in the tumor cells are considered as the oncogenic factors of these virus-positive Merkel cell carcinoma (VP-MCC). In contrast, the molecular pathogenesis of virus-negative MCC (VN-MCC) is less well understood. Using gene expression analysis of MCC cell lines, we found histone methyltransferase PRDM8 to be elevated in VN-MCC. This finding was confirmed by immunohistochemical analysis of MCC tumors, revealing that increased PRDM8 expression in VN-MCC is also associated with increased H3K9 methylation. CRISPR-mediated silencing of PRDM8 in MCC cells further supported the histone methylating role of this protein in VN-MCC. We also identified miR-20a-5p as a negative regulator of PRDM8. Taken together, our findings provide insights into the role of PRDM8 as a histone methyltransferase in VN-MCC tumorigenesis. KW - histone KW - histone methyltransferase PRDM8 KW - chromatin regulator KW - EGR1 KW - miRNA KW - MCV-negative KW - Merkel cell carcinoma KW - MCPyV Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203815 SN - 2072-6694 VL - 12 IS - 4 ER - TY - JOUR A1 - Orouji, Elias A1 - Peitsch, Wiebke K. A1 - Orouji, Azadeh A1 - Houben, Roland A1 - Utikal, Jochen T1 - Oncogenic role of an epigenetic reader of m\(^6\)A RNA modification: YTHDF1 in Merkel cell carcinoma JF - Cancers N2 - Merkel cell carcinoma is a deadly skin cancer, which in the majority of cases is caused by the Merkel cell polyomavirus (MCPyV). The viral small T antigen is regarded as the dominant oncoprotein expressed in the tumor cells. We used genomic screening of copy number aberrations along with transcriptomic analysis to investigate regions with amplification that harbor differentially expressed genes. We identified YTHDF1, a protein that is a reader of N\(^6\)-methyladenosine (m\(^6\)A) RNA modifications, to have high copy gains and to be highly expressed in Merkel cell carcinoma. Importantly, we identified the presence of m\(^6\)A on small T antigen mRNA suggesting a relation between YTHDF1 amplification and MCPyV gene expression. Interestingly, knockdown of YTHDF1 in Merkel cell carcinoma (MCC) cell lines negatively affected the translation initiation factor eIF3 and reduced proliferation and clonogenic capacity in vitro. Furthermore, analysis of survival data revealed worse overall survival in YTHDF1\(^{high}\) MCC patients compared to YTHDF1\(^{low}\) patients. Our findings indicate a novel oncogenic role of YTHDF1 through m\(^6\)A machinery in the tumorigenesis of MCC. KW - Merkel cell carcinoma KW - Merkel cell polyomavirus KW - copy number variations KW - m\(^6\)A KW - RNA modification KW - epitranscriptome KW - YTHDF1 KW - epigenetic reader Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200815 SN - 2072-6694 VL - 12 IS - 1 ER - TY - JOUR A1 - Fan, Kaiji A1 - Zebisch, Armin A1 - Horny, Kai A1 - Schrama, David A1 - Becker, Jürgen C. T1 - Highly expressed miR-375 is not an intracellular oncogene in Merkel cell polyomavirus-associated Merkel cell carcinoma JF - Cancers N2 - miR-375 is a highly abundant miRNA in Merkel cell carcinoma (MCC). In other cancers, it acts as either a tumor suppressor or oncogene. While free-circulating miR-375 serves as a surrogate marker for tumor burden in patients with advanced MCC, its function within MCC cells has not been established. Nearly complete miR-375 knockdown in MCC cell lines was achieved using antagomiRs via nucleofection. The cell viability, growth characteristics, and morphology were not altered by this knockdown. miR-375 target genes and related signaling pathways were determined using Encyclopedia of RNA Interactomes (ENCORI) revealing Hippo signaling and epithelial to mesenchymal transition (EMT)-related genes likely to be regulated. Therefore, their expression was analyzed by multiplexed qRT-PCR after miR-375 knockdown, demonstrating only a limited change in expression. In summary, highly effective miR-375 knockdown in classical MCC cell lines did not significantly change the cell viability, morphology, or oncogenic signaling pathways. These observations render miR-375 an unlikely intracellular oncogene in MCC cells, thus suggesting that likely functions of miR-375 for the intercellular communication of MCC should be addressed. KW - miR-375 KW - antagomiRs KW - Merkel cell carcinoma KW - Hippo signaling KW - focal adhesion Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200678 SN - 2072-6694 VL - 12 IS - 3 ER -