Pharmacological disruption of the microtubule array to determine the role of Golgi-nucleated microtubules in cancer cell migration

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Breast cancer is an increasingly common disease, with 1 in 10 women in the United States receiving a diagnosis of the disease during their lifetime. Taken alone, cancer of the breast is not particularly dangerous, however the tendency of this cancer to metastasize and move to other areas of the body greatly increase its mortality rate. The availability of breast cancer cell lines with varying migratory behavior make these cells useful models for studying molecular mechanisms of cellular motility. One mechanism hypothesized to contribute to this motility has been increasingly studied in the past two decades. The discovery of a specific subset of microtubules in the cell which originate, or nucleate, at the Golgi have been the focus of this research. These structures have been linked to the transport of signaling molecules and proteins which interact with the extra cellular matrix. Due to their ability to polarize, these microtubules can deliver intracellular cargo directly to the leading edge of the cell, implicating them in directed cell migration. Recent work by Laura Zahn in our lab has revealed an increased presence of these microtubules nucleated at the Golgi in cells with aggressive migratory behavior and cells treated with the chemotactic epidermal growth factor (EGF). Immunofluorescence microscopy was utilized for these observations. The objective of this thesis was to continue testing this hypothesis by observing the role of these microtubules in cell migration. This was accomplished utilizing pharmacological agents targeting the Golgi and microtubules to determine their effects on migration with a scratch wound assay. A secondary objective of this study was to determine if the morphology of the cells or organization of the Golgi and microtubules within the cell correlated to migratory behavior. The migratory behavior of the less invasive MCF7 cell line was slightly reduced by the disruption of its Golgi with brefeldin-A (BFA) treatment, but not depolymerization of its microtubules with nocodazole treatment. However, the more invasive MDA-MB-231 cell line was significantly impacted by both of these disruptions. Supplementation with the chemotactic factor EGF had mixed effects. This treatment returned Golgi-disrupted migratory behaviors to levels nearly that of control cells. However, this treatment was unable to rescue the cells from their inhibited migratory behavior caused by global microtubule depolymerization induced by nocodazole treatment. The morphology of the MDA-MB-231 cells was also affected by disruption of these structures. Treatment with either pharmacological reagent caused cells to lose adhesion to the matrix beneath the cells, causing them to round. This change in morphology, which was rescued with EGF supplementation, is associated with a significant reduction in migratory behavior The results of this study build support for the current hypothesis that the Golgi-nucleated subset of microtubules play a critical role in directed cell migration, especially in a highly invasive cell type. It is likely that the disruption of these structures prevents polarized delivery of intracellular cargo, including the EGF receptor, affecting directional migration.

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