Die Bedeutung der Formine FMNL1, 2 und 3 für die Invasivität und das Wachstum von Melanomzellen in vitro und in vivo

Aktinfilamente sind ein wesentlicher Bestandteil des Zytoskeletts in Eukaryonten. Für zahlreiche essentielle zelluläre Prozesse wie die Migration, die Adhäsion, den intrazel-lulären Vesikeltransport und die Zytokinese ist der zielgerichtete, rasche Auf- und Abbau von Aktinfilamenten unerlässlich. Ei...

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Bibliographic Details
Main Author: Hauswald, Maria
Contributors: Czubayko, Frank (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
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Actin filaments are crucial components of the eukaryotic cytoskeleton. The rapid, specific assembly and disassembly of actin filaments is essential for various cellular processes, i.e. migration, adhesion, intracellular vesicle transport and cytokinesis. Differ-ent actin nucleators, such as the formins FMNL1-3, play a major role in regulating these processes. In many tumor diseases – such as malignant melanoma – metastasis is a main cause of death. Apart from the invasiveness of tumor cells, the capability of adhering to the extracellular matrix (ECM) is a crucial factor for tumor and metastatic growth. As both processes depend on the reorganization of the actin cytoskeleton, it is particularly interesting to investigate the role of formins in these events in human melanoma cells. Current research mainly focused on the influence of FMNL2 on cancer cell invasion. Thus, the topic of this thesis was to investigate the effect of FMNL1-3 on cancer cell invasion and furthermore on cellular proliferation. Using siRNA- and shRNA- mediated knockdown of FMNL1-3 in the two invasive melanoma cell lines WM278 and LOX, we show that mainly FMNL2 influences the invasiveness of cells. Furthermore, FMNL2 and especially FMNL3 are necessary for anchorage-independent growth in vitro. Additionally, we demonstrate in mouse studies an effect of FMNL2 and FMNL3 on tumor initiation and growth of MV3 and LOX cells. Further analysis of these cells showed a deficiency in cell cycle progression due to an impaired G1- to S-phase transition resulting in an enhanced percentage of cells in G1. Moreover we could show an influence of FMNL2 and FMNL3 on the number of focal adhesions, subsequently indicating an impaired cell-matrix interaction. In each analy-sis, a combined knockdown of FMNL2 and FMNL3 displayed the strongest effects, indicative of a redundant function of these two formins. Thus, the concentration of both proteins has to deceed a certain limit in order to obtain strong effects as a de-crease in proliferation. This is likely due to a hetero-dimerization of FMNL2 and FMNL3, which we observed in co-immunoprecipitation and IP-mass spec studies. Thus, our data show that FMNL2 and FMNL3 play important roles for the prolifera-tion of melanoma cells in vitro and in vivo, in addition to their requirement for cancer cell invasion. Presumably, the reduced amount of focal adhesions after FMNL2- and/or FMNL3 -knockdown impairs the cell cycle leading to a reduced proliferation.