MIEN1 Regulates Breast Cancer Cell Migration and Invasion by Altering Cytoskeletal Dynamics Through Focal Adhesion Kinase and N-WASP Signaling

Purpose: Triple negative breast cancer (TNBC), accounts for approximately 15-20% of all breast cancer diagnoses. This is the most aggressive breast cancer subtype and is characterized by a lack of known receptors associated with, making prognosis and treatment difficult in patients with TNBC. TNBC has a propensity to metastasize to vital organs, including lung, brain and bone. This can occur early in the disease progression and usually leads to the elevated mortality rate in TNBC patients. Research efforts to identify molecular markers within TNBC for prognosis and therapy have not been fruitful. Migration and Invasion Enhancer 1 (MIEN1) has been implicated in the disease progression of many cancers, including TNBC. We determined to further understand the molecular mechanisms by which MIEN1 regulates cell motility and invasion in the context of TNBC. This knowledge will provide a basis to pursue MIEN1 as a potential marker for future treatment and evaluation of TNBC cases. Methods: Wild-type MIEN1 (MIEN1-WT) or Immunoreceptor tyrosine-based activation motif (ITAM)-mutant MIEN1 MIEN1-Y39/50F) was overexpressed in MDA-MB-231 cells to evaluate the role of ITAM signaling in MIEN1 mediated migration and invasion. Migration speed and persistence toward a chemoattractant was assessed using microfluidic chambers. Invasion was evaluated by embedding cell aggregates in a 3D collagen matrix and examining the spread of the cells. MIEN1 influence on migration was mediated by actin cytoskeletal dynamics. This mechanism was further delineated by looking at actin polymerization as well as focal adhesion adaptors and signaling molecules using western blotting as well as confocal microscopy. An in vitro kinase assay was also used to evaluate activators of MIEN1. Results: MIEN1-WT over-expression in MDA-MB-231 cells resulted increased migratory and invasive capabilities compared to wild-type cells. Additionally, over-expression of the MIEN1-Y39/50F ITAM mutant inhibited the cells’ ability to migrate towards a chemoattractant as well as invade through a collagen matrix. MIEN1 co-localized to the cell membrane with FAK (focal adhesion kinase) and facilitated signaling through N-WASP to alter cytoskeletal dynamics and increase filamentous actin accumulation. Conclusion: MIEN1 regulates migration and invasion of TNBC cells by altering cytoskeletal dynamics through activation of FAK and N-WASP, which results in increased actin polymerization and cell motility.