Abstract 1878: MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions

The epithelial-to-mesenchymal transition (EMT), and its reverse process, the mesenchymal-to-epithelial transition (MET), are associated with metastatic tumor progression in breast cancer (BC). E-selectin, a cell adhesion molecule expressed by cytokine-activated vascular endothelium, is also involved in metastasis via binding to counter-receptors (E-selectin ligands) on the surface of BC cells. Consequently, efforts have been made to investigate a link between the EMT/MET and the adhesive interactions between E-selectin and their ligands. Our lab previously demonstrated that BC cells with an epithelial phenotype exhibit more adhesive interactions between E-selectin/ligands under blood flow conditions than cells with a mesenchymal phenotype. Therefore, molecular factors in the blood that activate the EMT or the MET in circulating tumor cells (CTCs) have been of particular interest. These factors may regulate the potential for CTCs to traffic to bone marrow endothelium, a frequent site of BC metastasis that constitutively expresses E-selectin. To advance knowledge in this area, we hypothesized that miRNA-200c, associated with poor prognosis in metastatic BC patients, induces the MET in MDA-MB-231 BC cells and promotes adhesion between E-selectin/ligands. Microscopy images show that MDA-MB-231 cells transfected with miRNA-200c displayed a cell morphology similar to that of epithelial cells, whereas cells transfected with a negative control miRNA (NC miRNA) retained their mesenchymal phenotype. Furthermore, cells transfected with miRNA-200c exhibited greater mRNA expression of E-cadherin and lower expression of Vimentin compared to cells transfected with a NC miRNA by qRT-PCR. The expression of Zeb1/2, both targets for miRNA-200c, were down-regulated in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Both the microscopy images and mRNA expression analysis indicate induction of MET in miRNA-200c transfected cells. Shear flow adhesion assays and shear flow detachment assays were performed at bone marrow shear stresses to examine adhesive interactions between E-selectin/ligands. Using these assays, MDA-MB-231 cells transfected with miRNA-200c demonstrated slower rolling velocities, greater firm adhesion, lower detachment, and equivalent tethering activity when compared to cells transfected with a NC miRNA. Moreover, analysis of the mRNA expression of α1,3- and α1,4- fucosyltransferases (FT) revealed increased expression of FT3 and FT6 in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Altogether, these results suggest that miRNA-200c increases fluid-shear resistant adhesion and that factors present in the blood may impact the metastatic potential of cancer cells in circulation by regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1878.