AHNAK context-dependently regulates breast cancer cell mechanics, endothelial adhesion, and survival under fluid shear stress.

Breast cancer metastasis remains the leading cause of disease-related mortality, yet the molecular mechanisms enabling tumor cells to survive circulation and colonize distant organs are incompletely understood. AHNAK, a large scaffold protein implicated in cytoskeletal organization and membrane dynamics, has been reported to exert context-dependent roles in cancer progression, but its contribution to metastatic traits in less aggressive breast cancer cells and under biomechanical stress remains unclear. In this study, we investigated AHNAK expression and function across breast cancer cell models, with a particular focus on its role in modulating cellular mechanics, adhesion, and survival under fluid shear stress (FSS). We first compared AHNAK expression in MDA-MB-231, MDA-MB-231-Br, MCF-7 wild-type, and AHNAK-overexpressing MCF-7 cells, revealing differential expression patterns. Functional analyses demonstrated that AHNAK overexpression in MCF-7 cells did not significantly alter migratory behavior or morphology but increased cellular stiffness, as measured by atomic force microscopy, and increased clonogenic capacity. Conversely, AHNAK knockdown led to increased clonogenic capacity in MDA-MB-231 cells, indicating that the functional effects of AHNAK may vary according to the molecular and phenotypic characteristics of breast cancer cells. AHNAK overexpression also enhanced adhesion of MCF-7 cells to human brain microvascular endothelial cells as well as elevated ICAM-1 expression. Using an in vitro circulation model, we further show that exposure to physiologically relevant levels of FSS reduced overall cell survival; however, AHNAK-overexpressing cells consistently displayed a trend toward increased resistance to shear-induced cell death, with significant changes in post-flow adhesion to endothelial cells. Collectively, these findings indicate that AHNAK exerts cell-type-dependent effects on breast cancer cell phenotypes, promoting increased cellular stiffness, endothelial adhesion, and shear resistance in MCF-7 cells, while its depletion enhances clonogenic capacity in MDA-MB-231 cells. These findings underscore the importance of cellular context when considering the contribution of AHNAK to breast cancer progression and metastatic traits.