The tumor suppressor MYPOP: Characterization of novel MYPOP antibodies and MYPOP application as inhibitor of cell proliferation
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Abstract
Globally, human papillomaviruses (HPV) are the most common sexual infection (Plotzker et al., 2023), affecting over 80 out of 100 people in sexually active populations over the course of their life. Infections with high-risk HPV types imply a cancer risk due to viral oncogenes that transform infected cells. Despite the development of vaccines, HPV still account for 4.5% of the global cancer burden, such as oropharyngeal, cervical, and different types of skin cancers. Given that cancer is the second leading cause of death worldwide and HPV plays a significant role in its development, the importance of research on HPV and cancer is undeniable. Generally, cancer onset is attributed above all to genetic mutations. These mutations result in activated oncogenes or non-functional tumor suppressor proteins, enabling cells to divide uncontrollably. One of these tumor suppressor proteins is MYPOP, an interaction partner of the smaller capsid protein L2 of HPV (Wüstenhagen et al., 2018). MYPOP also binds HPV DNA, suppresses viral transcription, and thus exerts a protective effect in the human cell. Various normal cell lines have a well detectable amount of MYPOP. However, it is not detectable in immortalized or tumor cells of different cancer types. Moreover, overexpression of MYPOP in these tumor cells through transfection has been shown to provoke reduction of cell counts (Wüstenhagen et al., 2018). Due to the recent research on MYPOP, the knowledge in the literature is very limited and further research approaches are needed. For this reason, the objective of this project was to test new MYPOP antibodies and their application as markers of healthy tissue and explore ways of using MYPOP for potential targeted cancer therapy.
First monoclonal MYPOP antibodies were tested on normal skin cells, immortalized cells, and cervical cancer cells. In these experiments, three antibodies were identified that reliably differentiate between normal and immortalized cells based on MYPOP expression, which implies a potential use in diagnostics. Compared to the available polyclonal MYPOP antibodies, at least equivalent reliability in Western blot (WB) analyses was observed.
In the search for appropriate applications of MYPOP with the aim of reducing growth of cancer cells, directly applied purified MYPOP protein appeared to exhibit an inhibitory effect, which warrants further detailed investigation. For the first time, MYPOP was attempted to be applied as vector-based gene therapy in vitro using HPV16 pseudovirus (PsV). Initially, a successful inhibitory effect was suspected, but it turned out to be a phenomenon rather attributable to the PsV themselves. As the observed cytotoxic effect occurred only in tumor and not in healthy cells, the results still advocate for further exploration. The inclusion of recent findings of the Florin group leads to the assumption that the MYPOP protein itself poses challenges in the production of the PsV. Due to its antiviral properties, it likely interacts with capsid proteins or the DNA to be packaged and prevents efficient packaging or transcription of the expression
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plasmid. This issue should be considered and addressed in the future production of MYPOP PsV.
In summary, this work identifies suitable monoclonal MYPOP antibodies for future production and application in research and potential diagnostics. In addition, this project contributes to the path towards a potential therapeutic application of MYPOP in cancer therapy, testing vector- based application and highlighting issues to be considered in MYPOP-vector preparation.