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Information × Registration Number 0219U000058, 0118U005080 , R & D reports Title Molecular mechanisms of tumor cell plasticity controlling epithelial-mesenchymal/mesenchymal-amoeboid/mesenchymal-epithelial transitions (EMT/MAT/MET): the role of adaptor protein Ruk/CIN85. popup.stage_title Head Horak Iryna Romanivna, Registration Date 10-01-2019 Organization A.V.Palladin Institute of Biochemistry of National Academy of Sciences popup.description2 The object of the study is 4T1 murine breast adenocarcinoma cells with stable overexpression and downregulation of adaptor protein Ruk/CIN85. Purpose: to investigate the possible molecular mechanisms mediating the involvement of adaptor protein Ruk/CIN85 in the control of 4T1 breast adenocarcinoma cells plasticity, associated with changes in cell motility, invasiveness and metastasis. Methods of research: molecular biology, biochemical, optical, statistical. mRNA expression of the genes involved in the epithelial-mesenchymal transition (EMT) was analyzed by quantitative PCR in the 4T1 cells with overexpression and downregulation of adaptor protein Ruk/CIN85 in comparison with the control sublines. It was demonstrated that overexpression of Ruk/CIN85 in 4T1 cells is accompanied by a significant increase in the expression of Zeb1, Zeb2, Snai1, Twist1, Vim, Il1a, Il6, Cxcl5, Plau, Serpine1, Lox genes mRNA, and a significant inhibition of mRNA expression of the KlF17, Myb, Cdh1, Tjp1, Icam1, Itgb1, Cfl1, Cl2, Tnf, Mmp2, Mmp9, Ctsd, Plaur compared to control. Downregulation of Ruk/CIN85 leads to a significant increase in the expression of Klf17, Myb, Icam1, Itgb1, Cfl1, Clcl2, Tnf, Mmp2, Mmp9, Ctsd, Plaur and a significant reduction in the expression of Zeb1, Zeb2, Snai1, Twist1, Vim, Il1a, Il6, Cxcl5, Mmp14, Plau, Serpine1, Lox, Loxl2 compared to control sublines. The analysis of the activity of enzymes involved in the remodeling of the extracellular matrix demonstrated that the activity of matrix metalloproteinases of MMP-2 and MMP-9 and polyaminooxidase is significantly lower, while the activity of diamine oxidase and semicarbazide-sensitive amine oxidase increase in the conditioned medium produced by Ruk/CIN85-overexpressed 4T1 cells. Downregulation of Ruk/CIN85, on the contrary, leads to the growth of MMP-2, MMP-9 and polyaminooxidase activity in comparison to control. The expression levels of the genes involved in the reprogramming (Klf4, Pou5f1/Oct4, Sox2, Myc, Nanog) and surface markers of cancer stem cells (Cd24 and Cd44) in 4T1 cells with different Ruk/CIN85 content were estimated by quantitative PCR. It was shown that Ruk/CIN85-overexpressing 4T1 cells are characterized by significantly increased expression of Klf4, Nanog and Cd44, and decreased expression of Cd24, while Ruk/CIN85 downregulation leads to a significant decrease in the expression levels of Pou5f1/Oct4, Cd44, and increase in the Cd24 expression. Also, Ruk/CIN85 overexpression was accompanied by an increased ability of 4T1 cells to form spheroids (mammospheres), and Ruk/CIN85 downregulation led to the loss of this ability, indicating the potential role of Ruk/CIN85 in the control of cancer stem cells features. The obtained results indicate that Ruk/CIN85-overexpressing 4T1 cells are characterized by acquiring epithelial-mesenchymal/mesenchymal-amoeboid plasticity, while downregulation of Ruk/CIN85 leads to maintaining 4T1 cells in the epithelial phenotype. Product Description popup.authors Геращенко Денис Сергійович Скатерна Тетяна Дмитрівна popup.nrat_date 2020-04-02 Close