Roles of Semaphorin and Plexin Proteins on programmed cell death Protein 1 inhibitor resistance in Melanoma

Abstract

Melanoma is a common skin tumor with high mortality, especially in Europe, North America, and Oceania countries. Immunotherapy plays an important role in the treatment of melanoma. In the immunotherapy of melanoma, immune checkpoint inhibitors (ICI), such as PD-1 inhibitors, have been shown their significant therapeutic effects on malignant melanoma. However, many patients do not respond to immune checkpoint inhibitors, even emerged resistance. There are many reasons for ICI ineffectiveness or resistance, but it is still not fully understood at present. Therefore, the search for relative genes that may cause ICI tolerance has become a hot spot for further studies. Sema4D is also known as CD100. It expresses on cell surface of T-cells and many cancers. When Sema4D binds to its receptor Plexin-B1, they will play crucial roles in the immune regulation, angiogenesis, and tumor progression. However, the roles of Sema4D in melanoma and anti-PD-1 resistance are still unknown. Thus, in this research, the roles of Sema4D, Plexin-B1, and the relevant mechanism were explored on anti-PD-1 resistance in melanoma. To explore the roles of Sema4D and Plexin-B1 on anti-PD-1 resistance in melanoma, combination of molecular biology techniques and bioinformatics was used. The mRNA levels and protein expressions were detected by qRT-PCR and Western blotting. Cell viability and apoptosis were detected by CCK-8 and flow cytometer, cell invasion and migration were detected by matrigel invasion assay and wound healing, respectively. In vivo, Balb/c mice were used to detect the tumor volume and tumor weight. The results showed that the mRNA and PD-L1 protein expressions, Sema4D, and PlexinB1 in B16-F10 (murine melanoma) and SK-MEL-28 cell lines (human melanoma) were significantly decreased when compared with B16-F10R cell line (anti-PD-1 resistance murine melanoma). When Sema4D was knocked down and anti-PD-1 was cotreated, the cell viability, cell invasion, and migration of B16-F10R-Sema4D-shRNA cells were significantly decreased, while the apoptosis was increased. Mechanistically, bioinformatics analysis revealed that Sema4D is involved in the PI3K-AKT signaling pathway. Downregulation of p-PI3K/PI3K and pAKT/AKT expressions were observed in Sema4D knockdown group using shRNA. Additionally, tumor volume and tumor weight were also inhibited significantly when compared with B16-F10R-Sema4D-NC groups. In all, this research shown that Sema4D participates in regulating anti-PD-1 sensitivity in melanoma. Down regulation of Sema4D could increase the tumor apoptosis and attenuate PD-1 inhibitor resistance in melanoma. In conclusion, the results demonstrate that Sema4D deficiency potentiates anti-PD-1 treatment efficacy in melanoma cells via PI3K/AKT signaling pathway. Therefore, Sema4D may be a new indicator to influence anti-PD-1 sensitivity.