MMP9 drives cancer invasion by mediating integrin membrane trafficking and stabilization

Matrix stiffening promotes cancer progression by elevating matrix metalloprotease (MMP) activity, thereby enabling extracellular matrix degradation and migration. Yet, the nonproteolytic roles of MMPs in invasion remain less defined. Here, we dissect the proteolytic and nonproteolytic functions of MMP9, a metalloprotease whose expression and secretion increase sharply in highly invasive cancer cells in response to stiffness. We find that MMP9 supports cell spreading and 2D migration through a nonproteolytic mechanism that stabilizes focal adhesions, whereas its proteolytic activity is specifically required for 3D invasion. We further identify two distinct integrin β1 (ITGβ1) binding sites that coordinate these functions: The hemopexin domain drives copackaging and transport of the ITGβ1‐MMP9 complex to the cell periphery, while the RGD motif stabilizes ITGβ1 at the membrane before matrix degradation. These findings reveal a spatiotemporal mechanism by which MMP9 integrates adhesion dynamics with matrix remodeling to optimize invasive behavior.