Cancer metastasis requires tumor cell invasion, migration, and proliferation. Cell migration involves a complex series of processes that extend membrane protrusions to form substrate adhesions that cause cytoskeleton contraction within the cell. Further, cell migration is central to tissue repair, regeneration, development, cancer, and inflammation. The ferlins, an evolutionary conserved protein family, have been implicated as critical to maintaining plasma membranes. Specifically, myoferlin (MYOF), a mammalian ferlin, has been shown as responsible for membrane exocytosis/endocytosis and myoblast fusion. More recent studies have demonstrated that MYOF affects the anti-angiogenic response of endothelial cells and impacts the invasive ability of MDA-MB-231 breast cancer cells. It has also been noted that ablating MYOF using RNAi strategies results in consistent cell proliferation rates and a mesenchymal to epithelial phenotypic transformation.
In this study, we present quantified 2D morphologic and migration differences in MDA-MB-231 wild-type (231WT) and RNAi-mediated MYOF-deficient (231MYOFKD) human breast adenocarcinoma (MDA-MB-231) cells. Morphometrics found that MYOF deficiency led to significant differences in lamellipodia number and surface area, filopodia length, and cell surface area. These marked changes could suggest that MYOF plays a role in regulating cytoskeletal arrangement in breast cancer cells.
Prior studies used Boyden Chamber to determine MYOF’s effect on cell migration, which only provided path-independent data without demonstrating expected migration behavior based on MET. These limitations were overcome in this thesis using live-cell imaging platforms to quantify cell migration. The 2D in vitro wound assay showed a marked difference between 231 control (wild-type and lentiviral control) and 231MYOFKD cell size, cell velocity, and cell directionality during wound closure. The more unidirectional movement of 231MYOFKD cells mimicked that of epithelial cell migration, while the 231 control cells exhibited a more mesenchymal-like random migration pattern. Furthermore, 231MYOFKD cells exhibited a greater inclination towards spreading and cobblestone morphology reminiscent of the epithelial phenotype. On the other hand, the 231 control cells with a lack of cell-cell coordination and a more elongated cell shape resembled the mesenchymal phenotype.
It was also demonstrated through immunofluorescence that 231 control cells exhibited a stronger actin and focal adhesion site expression. In addition, atomic force microscopy (AFM) characterized cellular biomechanical properties and demonstrated that 231MYOFKD cells remodeled the actin cytoskeleton with less stress fibers and decreased cell stiffness (Young’s Modulus). Lastly, a cell motility polymerase chain reaction (PCR) kit provided potential genes affected by the MYOF deficiency—Calpain 1, Dipeptidyl Peptidase 4, Epidermal Growth Factor, Integrin Beta-2, and Myosin Heavy Chain 9.
Collectively, these studies demonstrated that MYOF depletion is associated with several notable outcomes: 1) change in cell protrusion characteristics, 2) change in cell behavior and phenotype, 3) change from a random to directional migration, 4) a decrease in cell stiffness due to cytoskeletal remodeling, and 5) a possible change in focal adhesion sites. Continued research on MYOF may provide insight into methods that may reduce metastasis and provide a more detailed rationale for the affects described in this study.