Semaphorin 5A modulates focal adhesion pathway and lamellipodia formation in melanoma

Metastatic potential and rising incidence make cutaneous melanoma a major health concern worldwide. Although the therapies currently available have proven effective, their use is limited by side effects arising and by the development of drug resistance. For this reason, research for new therapeutic targets and predictive biomarkers is crucial. Semaphorin 5A (SEMA5A) is a member of the semaphorin family with involvement in neural development and cancer biology. The laboratory where I completed my PhD has previously demonstrated in vitro that this protein promotes migration, invasion and, vasculogenic mimicry capability in melanoma cells. In this work, we further investigated the role and the underlying mechanism of action of SEMA5A in melanoma formation and progression. We performed a total proteome and in silico functional analysis using a stable SEMA5A knockdown model of A375 human melanoma cells, which showed that SEMA5A regulates pathways essential for cell viability, cell proliferation, and cytoskeletal dynamics, including focal adhesion and lamellipodia formation. We performed in vitro experiments to corroborate quantitative proteomic evidence by using both human and murine melanoma models. Results indicated that SEMA5A depletion affected clonogenic potential and cell viability under different culture conditions. By performing western blot analysis, we observed that SEMA5A silencing affected FAK and Paxillin phosphorylation, RhoA and Integrin β1 activation, thereby regulating focal adhesion dynamics and lamellipodia formation. In addition, it regulated the nucleation and expression of Arp3, the specific lamellipodia marker. Experiments conducted in vivo revealed delayed tumor formation and reduced tumor growth in mice injected with human melanoma cells expressing low levels of SEMA5A. These findings shed light on previously unrevealed functions of SEMA5A, promoting melanoma aggressiveness and progression, modulating cancer cell viability, focal adhesion signaling, and lamellipodia formation in vitro and tumor growth in vivo.