Introduction:
Metastatic potential and rising incidence make cutaneous melanoma a major health concern worldwide. While current therapies have proven effective, they are limited by side effects arising and by the development of drug resistance. For this reason, identifying new therapeutic targets and predictive biomarkers is crucial. Semaphorin 5 A (SEMA5A) is a member of the Semaphorin family, implicated in both cancer biology and neural development. We have previously demonstrated that this protein promotes different aggressive features of melanoma cells. In this study, we conducted a more in-depth investigation into the role of SEMA5A in the development and progression of melanoma.

Methods:
We performed proteomic and in silico analysis of stable knockdown SEMA5A human melanoma cells to identify the cellular pathways modulated by SEMA5A. The data used are available via ProteomeXchange with the identifier PXD065661. In vitro cell migration, clonogenic ability and cell viability assays under different adhesive substrate conditions were conducted using both human and murine melanoma SEMA5A-depleted clones. Western blotting and immunofluorescence analyses were employed to investigate focal adhesion regulation and lamellipodia formation. A Nocodazole assay was used to evaluate focal adhesion dynamics. A xenograft mouse model of melanoma was used to investigate the role of SEMA5A in tumor formation and tumor growth. Paraffin tumor sections were analyzed using immunohistochemical staining.

Results:
DAVID functional annotation tool revealed significant enrichment of Focal Adhesion and Integrin pathway-associated proteins in both control and SEMA5A-silenced cells. Ingenuity pathway analysis highlighted the role of SEMA5A in modulating several cellular pathways, including focal adhesion and lamellipodia organization. We demonstrated that SEMA5A depletion reduces the migration, clonogenic capacity, and viability of melanoma cells in both low and high stiffness culture substrates. SEMA5A was found to affect FAK and paxillin phosphorylation, as well as RhoA and Integrin β1 activation. This regulates focal adhesion dynamics, and lamellipodia formation. Furthermore, SEMA5A was found to regulate Arp3 nucleation and expression. Delayed tumor formation and reduced tumor growth were observed in mice bearing melanoma xenografts expressing low levels of SEMA5A. Immunohistochemical analysis of xenograft tumor sections revealed increased level of active Integrin β1 and phosphorylated FAK in SEMA5A-silenced tumors compared to control.

Conclusions:
These findings establish for the first time that SEMA5A is a novel player of melanoma aggressiveness and progression, modulating cancer cell viability and migration, focal adhesion signaling and lamellipodia formation in vitro and tumor growth in vivo.

Source:

Brignone, M., Cufaro, M.C., Ercolani, C. et al. Semaphorin 5A modulates focal adhesion pathway and lamellipodia formation in melanoma. Cell Commun Signal 23, 510 (2025). https://doi.org/10.1186/s12964-025-02526-z

https://link.springer.com/article/10.1186/s12964-025-02526-z