Methotrexate-modulated talin-dynamics drives cellular mechanical phenotypes via YAP signalling

Methotrexate is a well-known antineoplastic drug used to prevent cancer aggravation. Despite being a targeted therapeutic approach, its administration comes with the risk of cancer recurrence, plausibly through its proven off-target effect on focal adhesions. Since FA dynamics is dependent on force transmission through its constituent proteins, including talin, methotrexate might affect the mechanical activity of these proteins. Here we have combined single-molecule studies, computational dynamics, cell-based assays, and genomic analysis to unveil the focal adhesion-regulating role of methotrexate central to its effect on talin dynamics and downstream pathways. Interestingly, our single-molecule force spectroscopic study shows that methotrexate modulates the bimodal force distribution of talin in a concentration-dependent manner. Steered molecular dynamics reveal that methotrexate-talin interactions alter talin mechanical stability exposing their vinculin binding sites. Finally, we found that methotrexate-regulated talin-dynamics remodel cancer cell mechanical phenotypes like cell polarity, adhesion, and migration by regulating talin-vinculin association-mediated YAP signaling. These results further correlate with genomic analysis of methotrexate-treated patients, demonstrating its clinical importance. Taken together, these findings disseminate the effects of methotrexate-modulated mechanosensitivity of adhesion proteins on cellular events.