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The Epithelial-to-Mesenchymal Transition Regulates the E-selectin Ligand Activities of Breast Cancer Cells

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2016, Doctor of Philosophy (PhD), Ohio University, Chemical Engineering (Engineering and Technology).
During metastasis the epithelial-to-mesenchymal transition (EMT) increases the motility and invasiveness of breast cancer cells (BCs). However, it has yet to be determined whether the EMT affects the trafficking of BCs by regulating the expression of ligands that mediate adhesion with E-selectin presented by endothelial cells lining the blood vessel walls. Thus, the first aim of this investigation was to determine whether the capacity for BCs to mediate adhesion via E-selectin/ligand interactions is modified by the EMT. BCs within tissues from 110 cases of breast cancer were assayed for EMT biomarkers using immunohistochemistry and evaluated for E-selectin ligand activity using E-selectin microsphere adhesion assays. A significantly greater percentage of E-selectin microspheres adhered to BCs with an epithelial phenotype compared to BCs with a mesenchymal phenotype, indicating that the EMT is inversely correlated with E-selectin ligand activity. Similarly, BCs from cell lines with epithelial or mesenchymal phenotypes were assayed for E-selectin ligand activity using shear flow assays, in which significantly greater numbers of BCs with an epithelial phenotype adhered to E-selectin. Finally, the EMT was found to downregulate the E-selectin ligand activities of MDA-MB-468 cells and HMLE cells that underwent the EMT via ectopic expression of Snail or Twist, because in shear flow detachment assays greater percentages of cells with ectopic expression of Snail or Twist detached from E-selectin relative to the vector controls. Additionally, Hs578T cells that underwent the MET via shRNA knockdown of Snail or Twist demonstrated higher E-selectin ligand activities than the Hs578T shcontrol cells in detachment assays, as significantly greater percentages of Hs578T shSnail cells and Hs578T shTwist cells adhered to E-selectin compared to the Hs578T shcontrol cells. Interestingly, the adhesion of protease-treated cells to E-selectin was not significantly decreased by the EMT, indicating that the E-selectin ligand activities of glycoproteins and glycolipids were differentially affected by the EMT. Furthermore, qRT-PCR revealed that relative changes in gene expression for fucosyltransferases (FT) FT3 and FT6, which functionalize E-selectin ligands via the addition of fucose, paralleled the EMT or MET induced changes in the E-selectin ligand activities of BCs. The second aim of this investigation was to determine how the constitutive expression of E-selectin by bone marrow endothelial cells may affect the growth of BCs that metastasize to the bone marrow. Stem-like BCs with a mesenchymal phenotype, i.e., Hs578T cells, formed mammosphere-like structures on E-selectin-coated plates and activated HUVEC. In contrast, non-stem-like BCs with an epithelial phenotype were unaffected by E-selectin. RT2 PCR-profiler arrays and annexin V / propidium iodide staining indicated that Hs578T cells cultured on E-selectin pursued necrotic cell death via signaling mechanisms in which BCL-2 genes and TNF-receptor superfamily genes were expressed. Furthermore, RT2 PCR-profiler arrays implicated Notch and Wnt signaling in regulating the behaviors of Hs578T cells grown on E-selectin. The third aim of this study was to evaluate whether the apparent cellular mechanical properties of BCs are affected by the MET. A microfluidic device was used to deform the BCs and the apparent mechanical properties of BCs were calculated using a modified-power-law model. Compared to the Hs578T shcontrol cells, Hs578T shTwist cells were found to be more viscous, indicating that the MET increased the mechanical strength of BCs. In summation, this study provides a mechanism for the regulation of the E-selectin ligand activities of BCs via the EMT and MET, reveals how E-selectin may affect the growth of BCs in the bone marrow, and shows that the apparent cellular mechanical properties of BCs are strengthened by the MET.
Monica Burdick, PhD (Advisor)
168 p.

Recommended Citations

Citations

  • Carlson, G. E. (2016). The Epithelial-to-Mesenchymal Transition Regulates the E-selectin Ligand Activities of Breast Cancer Cells [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1477670221426081

    APA Style (7th edition)

  • Carlson, Grady. The Epithelial-to-Mesenchymal Transition Regulates the E-selectin Ligand Activities of Breast Cancer Cells . 2016. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1477670221426081.

    MLA Style (8th edition)

  • Carlson, Grady. "The Epithelial-to-Mesenchymal Transition Regulates the E-selectin Ligand Activities of Breast Cancer Cells ." Doctoral dissertation, Ohio University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1477670221426081

    Chicago Manual of Style (17th edition)