@phdthesis{Sarma2021,
  author    = {Sarma, Bhavishya},
  title     = {Merkel Cell Carcinoma: Investigations on its carcinogenesis and new therapeutic approaches},
  doi       = {10.25972/OPUS-24740},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247402},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with an increasing incidence. The majority of MCC cases (approximately 80\%) are associated with the Merkel cell polyomavirus (MCPyV). This virus encodes for the MCPyV T antigens (small T (sT) and large T (LT)), which are oncoproteins that drive MCC carcinogenesis. However, the precise cells of the skin that are transformed by the T antigens are not known i.e., the cells of origin of MCC are yet to be discovered. Therefore, the first part of this study involved the generation and evaluation of a vector system that could be used to study MCC oncogenesis. To this end, a set of lentiviral vectors was cloned that allows independent, inducible expression of potential key factors in MCC oncogenesis. In addition, a CRISPR/Cas9 knock in was established that allows the coding sequence for a fluorescent protein to be placed under the control of the promoter of KRT20, one of the most crucial markers of MCC. The functionality of this KRT20 reporter was proven in the MCPyV-positive MCC cell line, WaGa. The different inducible vector systems (doxycycline-inducible MCPyV T antigens or MCPyV sT, RheoSwitch-inducible ATOH1 and IPTG-inducible dnMAML1 and GLI1) were found to have different efficacies in various cellular systems and in particular, a considerable reduction in efficiency was observed at times upon the interaction of several vectors in one cell. In the second and more important part of this study, the role of the well-established anti-malarial drug, artesunate, which possesses additional anti-tumor and anti-viral activity, in the treatment of MCPyV-positive MCC was analyzed. In our study, artesunate was found to be cytotoxic towards MCPyV-positive MCC cell lines in vitro and repressed tumor growth in vivo in a mouse model. Artesunate was also found to downregulate T antigen expression, which is critical for the proliferation of MCPyV-positive MCC cells. The repression of T antigen expression, however, was not the sole mechanism of artesunate's cytotoxic action; instead, the MCPyV-positive MCC cell line, WaGa, was found to be even less sensitive to artesunate after shRNA knockdown of the T antigens. Since loss of membrane integrity occurred more rapidly than degradation/loss of genomic DNA under the influence of artesunate in four of five MCPyV-positive MCC cell lines examined, apoptosis, although widely described as a modus operandi for artesunate, did not appear to be a determinant of the cytotoxicity of artesunate against MCPyV-positive MCC cells. Instead, we were able to demonstrate that artesunate induced the recently described iron-dependent and lipid peroxide-associated form of cell death known as "ferroptosis". This was achieved primarily through the use of inhibitors that can suppress specific individual steps of the ferroptotic process. Thus, artesunate-induced cell death of MCPyV-positive MCC cells could be suppressed by iron chelators and by the inhibition of lipid peroxidation and lysosomal transport. Surprising results were obtained from the analysis of two proteins associated with the ferroptotic process, namely, ferroptosis suppressor protein 1 (FSP1) and tumor suppressor protein p53. Here, we showed that ectopically- 2 expressed FSP1 cannot suppress artesunate-induced ferroptosis in MCPyV-positive MCC cells and that p53 does not play a pro-ferroptotic role in artesunate-induced cell death of MCPyV-positive MCCs. Since artesunate did not suppress the interferon-γ-induced expression of immune-related molecules such as HLA and PD-L1 on the surface of MCPyV-positive MCCs, our study also provided the first positive evidence for its use in combinatorial immunotherapy. Overall, this study showed that artesunate appears to be an effective drug for the treatment of MCPyV-positive MCC and might also be considered for its use in combinatorial MCC immunotherapy in the future.},
  language  = {en}
}