Sema4D silencing increases the sensitivity of nivolumab to B16-F10 resistant melanoma via inhibiting the PI3K/AKT signaling pathway

Abstract

Melanoma is a common skin tumor that causes a high rate of mortality, especially in Europe, North America and Oceania. Immunosuppressants such as anti-PD-1 have been used in the treatment of malignant melanoma, however, nearly 60% of patients do not respond to these treatments. Sema4D, also called CD100, is expressed in T cells and tumor tissues. Sema4D and its receptor, Plexin-B1, play crucial roles in the process of immune regulation, angiogenesis, and tumor progression. The role of Sema4D in melanoma with anti-PD-1 resistance is poorly understood. Through a combination of molecular biology techniques and in silico analysis, the role of Sema4D in improving anti-PD-L1 sensitivity in melanoma was explored. The results showed that the expression of Sema4D, Plexin-B1 and PD-L1 was significantly increased in B16-F10R cells. Sema4D knockdown synergizes with anti-PD-1 treatment, cell viability, cell invasion and migration were significantly decreased, while the apoptosis was increased, the growth of tumors on the mice was also inhibited. Mechanistically, bioinformatics analysis revealed that Sema4D is involved in the PI3K/AKT signaling pathway; the downregulation of p-PI3K/PI3K and p-AKT/AKT expression were observed in Sema4D knockdown, therefore, nivolumab resistance is related to Sema4D and Sema4D silencing can improve sensitivity to nivolumab via inhibition of the PI3K/AKT signaling pathway.

Keywords: Melanoma; Nivolumab; PD-1 inhibitor resistance; PI3K/AKT; Sema4D.

Figure 1. Sema4D, Plexin-B1 and PD-L1 expression in B16-F10 and B16-F10R cells.

Cells were derived into B16-F10R group (B16-F10R cells) and B16-F10 (B16-F10 cells), 24 h after cell culture, Sema4D, Plexin-B1 and PD-L1 expression were detected. (A) Protein bands of Sema4D, Plexin-B1 and PD-L1. (B) Ratio to GAPDH of Sema4D. Image J V1.8.0 software was used to measure the gray value of the value of the protein band, and then the relative density value was obtained by comparing the value of GAPDH with the target protein, p = 0.0026. (C) Ratio to GAPDH of Plexin-B1, p value = 0.0018. (D) Ratio to GAPDH of PD-L1, p value = 0.0016. (E) mRNA expression of Sema4D, p value = 0.037. (F) mRNA expression of Plexin-B1 , p = 0.0312. (G) mRNA expression of PD-L1, p = 0.0212. Compared with B16-F10 group, Sema4D and Plexin-B1 were significantly upregulated in B16-F10R group. ^∗ is p < 0.05, ^∗∗ is p < 0.01, ^∗∗∗ is p < 0.001. Repeat the experiment three times (n = 3), t test was used for statistical analysis.

Figure 2. Sema4D silencing decrease PD-L1 expression.

B16-F10R Cell were derived into Sema4D-shRNA group and Sema4D-NC group, co-cultured with TIL at a 1:5 ratio and nivolumab 50 ng/ml 24 h, collect the cell to detect the expression of PD-L1. (A) Correlation between PD-L1 and Sema4D (B) Protein bands of PD-L1and Sema4D. (C) Ratio to GAPDH of Sema4D, p = 0.0002. (D) Ratio to GAPDH of PD-L1, p = 0.0016. (E) mRNA expression of PD-L1. Compared with Sema4D-NC group, ^∗∗ is p < 0.01, ^∗∗∗ is p < 0.001. The experiment was repeated three times, n = 3, t test was used for statistical analysis.

Figure 3. Sema4D repression renders B16-F10R cells sensitive to nivolumab treatment.

(A) Effects of nivolumab on cell viability of B16-F10 and B16-F10R. B16-F10 cells were derived into B16-F10 and B16-F10+nivolumab group, B16-F10R cells were derived into B16-F10R and B16-F10R+nivolumab groups, B16-F10 and B16-F10R groups were as control group without treatment, B16-F10+nivolumab and B16-F10R+nivolumab groups were treatment with 50 ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio (Van den Berg et al., 2020). Compared with the B16-F10+nivolumab group, ^∗∗ is p < 0.01, n = 3, one-way ANOVA was used to compare the differences. (B) RNA expression of Sema4D overexpression. Cells were derived into B16-F10+LV-Sema4D and B16-F10+LV-NC group, compared with the LV-NC group, ** is p < 0.01, n = 3, t-test was used to compare the differences. (C) Effects of nivolumab on B16-F10 cell viability after Sema4D overexpression. B16-F10 cells were derived into B16-F10, B16-F10+nivolumab, B16-F10+LV-Sema4D and B16-F10+LV-NC group, B16-F10 is control group, another 3 groups were treatment with 50ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio, B16-F10+LV-Sema4D group was also treated with overexpression Sema4D, B16-F10+LV-NC group was also treated with Sema4D negative control. Compared with B16-F10 group, **** is p < 0.0001; Compared with B16-F10+LV-NC group, #### is p < 0.0001, n = 3, one-way ANOVA was used to compare the differences. (D) Effects of nivolumab on B16-F10R cell after Sema4D silencing. B16-F10R cells were derived into B16-F10R, B16-F10R+nivolumab, Sema4D-shRNA and Sema4D-NC group , B16-F10R is control group, another 3 groups were treatment with 50ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio, Sema4D-shRNA group was knockdown Sema4D, and Sema4D-NC group was Sema4D-shRNA negative control group. Compared with Sema4D-shRNA group, * is p < 0.05, ** is p < 0.01, n = 3, one-way ANOVA was used to compare the differences. (E) B16-F10R cell apoptosis rate after treatment with nivolumab. B16-F10R cells were derived into Sema4D-shRNA group and Sema4D-NC group, the treatment is the same as C. Cell apoptosis was detected byPI and Annexin V/Alexa Fluor 647 double staining, Alexa Fluor 647 was a fluorescent dye binding with Annexin V, reflecting early apoptosis of cells. Compared with Sema4D-NC group, ** is p < 0.01, n = 3, t-test was used to compare the differences. (F) Tumor volume of knockdown Sema4D with nivolumab treatment. Balb/C mice were derived into Sema4D-shRNA group (n = 6) and Sema4D-NC group(n = 5), B16-F10R-Sema4D-shRNA cells and B16-F10R-Sema4D-NC cells were intradermal injected into the right side of mice, then intraperitoneally injected nivolumab 10 mg/kg once at day 1, t-test was used to compare the differences. (G) Tumor weight of knockdown Sema4D with nivolumab, t-test was used to compare the differences. Compared with Sema4D-NC group, *** is p < 0.001, **** is p < 0.0001. (H) Mouse melanoma specimen. Major marks represent cm on the scale.

Figure 4. Sema4D deficiency potentiates nivolumab inhibitory effects on B16-F10R cell invasion and migration.

B16-F10R cells were derived into Sema4D-shRNA group and Sema4D-NC group, co-cultured with TIL at a 1:5 ratio and nivolumab 50 ng/ml, cell invasion and migration were detected. (A) Cell migration. Representative image of cell migration in the absence and presence of shRNA Sema4D at 0 h, 24 h, and 48 h. The scratched areas were measured in three random fields in each group. Wound healing analysis showed a significant difference in the cell-free area of Sema4D-shRNA group was significantly wider than that of Sema4D-NC group at 24 and 48 h (P < 0.0001). (B) Ratio of cell free area. (C) Chamber invasion of cells. Chamber invasion analysis showed a significant difference lower number of invasive cells in Sema4D-shRNA group than Sema4D-NC group (× 100) at 48 h. (D) cell invasion number per field. Compared with Sema4D-NC group,^∗∗∗∗ is p < 0.0001. The expression was repeated three times, n = 3.

Figure 5. Sema4D knockdown is significantly associated with inhibition of PI3K-AKT signaling pathway.

(A) Association of clustered modules with Sema4D expression. The brown module was identified as the most relevant module for Sema4D expression. (B) Module-trait associations. (C) Gene set variation analysis revealed several significantly dysregulation malignancies events between different Sema4D expression population. (D) Protein-protein interactions of Sema4D. (E) Pathway enrichment analysis showed that Sema4D is involved in the PI3K-AKT signaling pathway. (F) PI3K-AKT signaling pathway proteins expression after Sema4D knockdown. B16-F10R cells were derived into Sema4D-shRNA group and Sema4D-NC group, co-cultured with TIL at a 1:5 ratio and nivolumab 50 ng/ml, PI3K, AKT protein expression were detected. The experiment was repeated three times, n = 3. Western blotting showed that Sema4D was significantly associated with inhibition of PI3K-AKT signaling pathway . Compared with Sema4D-NC group, ^∗∗ is p < 0.01.