Abstract 2840: Characterization of pazopanib resistance in renal cell carcinoma cell lines
Authors: Vynnytska-Myronovska Bozhena , Heinzelmann Joana , Hölters Sebastian , Stoeckle Michael , Junker Kerstin 
Published: August 2018

Abstract
BACKGROUND: Together with Sunitinib (Sun), Pazopanib (Paz) is considered as a gold standard in metastatic renal cell carcinoma treatment (RCC). Similarly to other tyrosine kinase inhibitors (TKI), development of resistance constitutes the main problem of Paz-based therapy. This warrants the in vivo investigations of the processes related to Paz resistance development.
METHODS: Parental human RCC cell lines, 786-O/wt and CAKI-2/wt, were continuously treated with Paz at IC50 concentration and Paz-resistant 786-O and CAKI-2 cell subpopulations (786-O/res and CAKI-2/res) were developed. Parental and resistant cells were compared in terms of the malignant phenotype by WST-1 proliferation, cross-resistance to another TKI, migration wound healing assays and signaling pathways analysis by Western blot. MiRNA expression was analyzed using microarrays (Agilent Technologies).
RESULTS: The in vitro analysis of RCC cells growth under Paz treatment revealed that this drug inhibited proliferation of parental 786-O and CAKI-2 cells with IC50 (inhibitory concentration of 50%) of 14 and 11 µmol/L, respectively. The continuous treatment with this drug resulted in the gradual resistance development in both tested cell lines. At the end of the period of continuous treatment (32 passages, 3 months) the resistance level of 786-O/res and CAKI-2/res cells was increased 2-fold compared with 786-O/wt and CAKI-2/wt. The analysis of cell motility revealed that the short-termed exposure to Paz retarded the migration of both parental and Paz-resistant 786-O and CAKI-2 cells. However, no differences in the motility rates of parental and resistant cells were observed. The analysis of the signaling pathways that might be involved in the resistance development revealed no changes in the expression of epithelial-to-mesenchymal transition (EMT) markers N-cadherin, vimentin and β-catenin between parental and Paz-resistant subpopulations of both cell lines. Cell response to Paz treatment differs between the tested parental RCC cell lines CAKI-2 and 786-O in terms of the expression of phosphorylated forms of the growth-promoting molecules of Akt-mTOR and MAPK-ERK pathways. A significant change in miRNA expression was found in both cell lines at different time points of treatment compared to parenteral cells.
CONCLUSIONS: Our data suggest that the process of Paz resistance development is cell line specific. Akt-mTOR and MAPK-ERK signaling pathways seem to be related to Paz resistance development in RCC. Development of resistance is associated with specific miRNA alterations. Further studies have to analyze the functional effects of miRNAs on resistance and possible signaling pathways.
Citation Format: Bozhena Vynnytska-Myronovska, Joana Heinzelmann, Sebastian Hölters, Michael Stoeckle, Kerstin Junker. Characterization of pazopanib resistance in renal cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2840.