Preview

PDF, English Download (5MB) | Terms of use

Abstract

Melanoma is a devastating disease with a growing incidence, which represents a challenge for scientists and physicians worldwide. Due to its high metastatic potential and the rapid development of resistances to established therapies, this cancer still accounts for the majority of skin cancer-related deaths. In my thesis, I could show that ADCK2 plays a role in melanoma cells, especially affecting their metastatic capacity. After knocking down ADCK2 I observed a lower cell viability, but better migration and invasion of melanoma cells. Moreover, I noticed a reduced pigmentation of the pellets of melanoma cell lines, which could be explained by lower expression of melanocyte and higher expression of neural crest cell (NCC) markers. These results indicate a phenotype switch of melanoma cells to a more de-differentiated state, which is often associated with a higher metastatic potential. Additional analyses revealed a positive correlation between ADCK2 and Sox5, FoxD3, MYL6 and RAB2A expression in melanoma cells. Since the ADCK2 knockdown (KD) most significantly affected the migrative capacity of melanoma cells, I continued investigating the connection with MYL6, a protein involved in cell motility. I could demonstrate that overexpression (OE) of ADCK2 in melanoma cells had the opposite effect on cell viability and motility compared to the KD. The effect of ADCK2 OE on cell viability and migration could be reversed by a KD of MYL6, which suggests that MYL6 could be a downstream effector of ADCK2. Immunofluorescence stainings of ADCK2 KD cells revealed that these cells showed an altered actin cytoskeleton compared to control cells. One can assume that MYL6 could influence the cell motility of melanoma cells due to its function within non-muscle myosin 2, a known binding partner of actin filaments. Since ADCK2 negatively affected the metastatic potential of melanoma cells, it could be considered a tumor suppressor. As tumorigenesis and development share many similarities, I further investigated the role of ADCK2 (and other kinases) in the differentiation process of human induced pluripotent stem cells (hiPSCs) towards neural crest cells (NCCs). First to mention, I generated and validated two hiPSC lines that were stably maintained on Matrigel, one by reprogramming fibroblasts and another one by subcloning an existing cell line. Both cell lines were then successfully differentiated into NCCs in a xeno-free environment in ten days. Moreover, I could detect a downregulation of stem cell and upregulation of NCC marker expression upon KD of ADCK2 and DBF4 on day 0 followed by ten days of differentiation. Due to this result, one can speculate, that a KD of ADCK2 and DBF4 could lead to a more efficient differentiation of hiPSCs into NCCs. However, additional experiments are necessary to test this hypothesis. Moreover, it needs to be examined if these NCCs are functional and resemble naturally occurring NCCs. Additionally, I could demonstrate that a KD of TYK2 or EPHA4 resulted in increased stem cell marker and decreased NCC marker expression, which indicates that these kinases might be necessary for NCC development. Further experiments are needed to clarify this assumption. To conclude, I found that ADCK2 plays a role in melanoma cell behavior, but no strong role in the development of NCCs. A KD of ADCK2 induced a phenotype switch of melanoma cells to a more NC-like identity, validated by an upregulation of NCC markers and an increased motility. It would be interesting to further study the involvement of ADCK2 in melanoma and NCC development, which could yield more insights into melanoma development and thereby could open up new possibilities for melanoma therapy or diagnosis.