Abstract 5066: Impact of Brk-inhibitors on nondirectional and directed migration of breast cancer cells

Abstract

Abstract The non-receptor tyrosine kinase Brk (breast tumor kinase, PTK-6) has been linked to various effects in tumor cells including cell proliferation, differentiation and migration. Modulation of cellular signaling occurs due to protein scaffolding and substrate phosphorylation, while effects depend on cell type and molecular context. Recent findings suggest, that some hallmark effects of cancer may be linked to the active kinase whereas inactive Brk seems to be involved in different physiological processes of epithelial cells. Novel α-carboline derivatives were described previously and identified as Brk inhibitors in in-vitro testing. We examined the effect of four candidates MK5, MK136, MK138 and MK150 in breast cancer cell lines regarding Brk as a modulator of cell migration. Cell lines MDA-MB-231 and MDA-MB-468 were cultivated as indicated by the distributor. Inhibition of non-directional migration over a given period of time was determined by scratch assays barring proliferation by addition of 50 mM hydroxyurea. The gap was created using silicon inserts and TScratch software was used to evaluate cell free surface area. Directional migration was investigated by trans-well assay utilizing FCS as attractant and an 8 μm pore membrane in combination with xCellingence real time measurement. Statistical evaluation was conducted using 1-way ANOVA followed by Dunnett's test. In scratch assays samples incubated with 1 μM of each substance showed a larger cell free area after 24 hours for MDA-MB-231 cells and after 30 hours for MDA-MB-46 cells. The increase was significant in MDA-MB-231 cells for substance MK136 (32.07±17.21%, p<0.1) and highly significant for MK138 (52.37±7.00%, p<0.001) compared to DMSO treated control (7.73±3.22%). Furthermore, the effect of MK138 increased in a concentration-dependent manner yielding consistently highly significant differences at a concentration of 1 μM (36.77±14.53%, N = 8 vs. 9.870±1.12%, N = 4). MK138 displayed a significant difference in MDA-MB-468 cells as well (37.27±12.56% vs. 10.07±3.04%, p<0.01). In transwell experiments MDA-MB-231 cells showed significantly impaired migration when incubated with MK138 and MK150. For MK138 a concentration-dependent effect was found similar to the scratch assay. In cell line MDA-MB-468 the inhibitor MK138 accounted for a significant decrease in directional migration as well. Our findings indicate an influence of pharmacological Brk-inhibition on the migratory ability of breast cancer cells. These effects were evident for all tested substances and constantly significant and concentration-dependent for MK138 making unspecific effects unlikely. The mechanism of Brk activity in modulation of migratory processes needs to be further addressed to better understand and exploit its influence on tumor cell migration, dissemination and metastasis. This work was supported by the German Research Foundation (DFG, Grant No. RI1196/4-1). Citation Format: Markus Oelze, Franziska I. Kluwe, Tom Wersig, Wolfgang Sippl, Andreas Hilgeroth, Christoph A. Ritter. Impact of Brk-inhibitors on nondirectional and directed migration of breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5066.