Leupaxin inhibits the durotaxis and mechanosensitivity of metastatic breast cancer cells

Efficient cancer cell migration is essential for invasion and metastasis and is driven by cancer cell interaction with their extracellular matrix (ECM). Thus, ECM properties determine the migration phenotype. For example, ECM stiffness can guide cancer cell migration through durotaxis; however, the mechanisms regulating cancer cell durotaxis remain poorly understood. Using in-house stiffness gradient hydrogels, we discovered that MDA-MB-231 breast cancer cell metastatic variants selected for either bone or brain organotropism display impaired durotaxis. Moreover, akin to cells conditioned on soft substrates, these cells have altered mechanoresponses to increasing stiffness, including reduced cell spreading and focal adhesion assembly and signaling. We observed upregulation of the LIM-domain protein leupaxin, a focal adhesion component, in these variants and in soft-conditioned MDA-MB-231 cells compared to parental cells. Leupaxin silencing in brain-tropic metastatic cells restored durotaxis, whereas its overexpression in parental cells impaired durotaxis and mechanosensing, phenocopying the metastatic variants. Mechanistically, leupaxin disrupted paxillin–focal adhesion kinase (FAK) signaling, thereby inhibiting durotaxis. Together, these findings identify leupaxin as a negative regulator of durotaxis in metastatic cells and suggest a mechanism by which tumor cells adapt to mechanical heterogeneity to facilitate metastatic progression.