The Extracellular Matrix Regulates Invasion in Fusion-Negative Rhabdomyosarcoma via YAP–PIEZO1 Signaling Axis

<b>Background</b><b>:</b> Fusion-negative rhabdomyosarcoma (FNRMS) represents the most prevalent subtype of rhabdomyosarcoma, the most common pediatric soft-tissue sarcoma. Although its invasion is a leading cause of recurrence and poor prognosis, its underlying mechanism remains unclear. We investigated how extracellular matrix density regulates FNRMS progression via mechano-transduction. <b>Methods:</b> We used three-dimensional spheroid invasion assays with FNRMS cells embedded in varying collagen concentrations. Mechanistic insights were gained through immunofluorescence, sequencing reanalysis, calcium live-cell imaging, and pharmacological inhibition of the YAP-PIEZO1 axis. <b>Results:</b> High extracellular matrix density significantly enhanced invasive spreading, correlating with increased YAP nuclear localization. YAP overexpression was sufficient to promote invasive spreading, while its inhibition attenuated the matrix-enhanced phenotype. We identified <i>PIEZO1</i> as a direct transcriptional target of YAP. High extracellular matrix density stimulated PIEZO1-dependent calcium influx, which was required for invasion. Furthermore, elevated <i>PIEZO1</i> expression was significantly associated with poorer overall survival in FNRMS patients. Targeting YAP effectively suppressed both calcium flux and invasion. <b>Conclusions:</b> Our findings establish a YAP-PIEZO1 axis linking extracellular matrix density to FNRMS invasion. This mechanosensitive pathway represents a potential therapeutic vulnerability in aggressive FNRMS.