Горак І. Role of adaptor protein Ruk/CIN85 in the control of migration and invasion of cancer cells in vitro and in vivo

This PhD thesis is aimed to investigate molecular mechanisms dependent on adaptor protein Ruk/CIN85 that control migration and invasion of cancer cells in vitro and in vivo. Murine breast adenocarcinoma 4T1 cells sublines with stable overexpression or knockdown of Ruk/CIN85 were generated. It was demonstrated that Ruk/CIN85 overexpression in sublines of 4T1 cells is accompanied by decreased proliferative potential and adhesiveness as well as increased motility and invasiveness in vitro, while Ruk/CIN85 downregulation leads to opposite effects. For the first time transcriptome analysis of cancer cells with different Ruk/CIN85 expression levels was performed. It revealed that adaptor protein Ruk/CIN85 modulates the expression of a number of genes involved in cancer cells migration and invasion, including transcription factors Twist1, Snai1, Zeb1/2, matrix metalloproteinases MMP-2, MMP-9, reprogramming factors Klf4, Oct4, Nanog. Ruk/CIN85 expression level in 4T1 cells correlates negatively with MMP-2 and MMP-9 activities. Activation of these enzymes inhibits invasion of Ruk/CIN85-downregulated cells via angiostatin production. The features of amoeboid-like motility in Ruk/CIN85-overexpressing 4T1 cells such as abrogated pericellular proteolysis, rounded cell shape, presense of cortical actin ring and membrane blebs, increased lysyl oxydase expression and activity were found. Ruk/CIN85 overexpression in 4T1 cells was demonstrated to potentiate cancer stem cells properties, such as anchorage-independent growth, self-renewal, expression of specific surface markers, doxorubicin resistance. It was found that overexpression of adaptor protein Ruk/CIN85 in 4T1 cells is accompanied by elevated extravasation and lung metastasis, while Ruk/CIN85 knockdown results in considerable quenching of metastatic potential. In the pulmonary metastases Ruk/CIN85 induces mesenchymal-epithelial transition, associated with enhanced proliferative activity of cancer cells. The data obtained indicate that Ruk/CIN85-overexpressing 4T1 cells are characterized by potentiation of epithelial-mesenchymal/mesenchymal-amoeboid plasticity, underlying increased cancer cells invasion and metastasis. Ruk/CIN85-downregulated cells lack such plasticity and acquire homogenous epithelial-like state.