Fig. 7. WSX1 relieves inhibition of GSK3β activity through impeding the PI3Kδ/AKT signaling pathway.

a Effect of WSX1 overexpression on protein levels of p-AKT^Ser473, AKT, p-TSC2^Thr1462, and TSC2 in HCC cells determined by immunoblotting. TSC2 is directly phosphorylated at Thr1462 by AKT; thus p-TSC2^Thr1462 levels indirectly reflect AKT activity. b Impact of AKT reactivation on WSX1-mediated PD-L1 downregulation detected by flow cytometry. SNU449 and SNU475 cells were transfected with WSX1 alone or co-transfected with membrane-bound myr-AKT. Statistical analysis results are shown on the right (n = 5 independent experiments, P < 0.0001 in both SNU449 and SNU475 cells). c Impact of myr-AKT transfection on protein levels of p-AKT^Ser473, total AKT, and PD-L1 in 449^WSX1 and 475 ^WSX1 cells. d Immunoblotting analysis of expression of WSX1, PD-L1, and p-AKT^Ser473 in whole liver lysates obtained from FVB/NJ mice injected with NRAS/AKT oncogenes with or without WSX1 (left, C1–C4 represent independent samples from “oncogene” group, T1–T4 are independent samples from “oncogene + WSX1” group). Statistical analysis of correlations of WSX1 with PD-L1 and p-AKT^Ser473 expression in mouse livers (right). e Effect of WSX1 on expression of PTEN, PI3K-p85, PI3K-p110α, and PI3K-p110δ. f Impact of PIK3CD overexpression on WSX1-mediated PD-L1 reduction (left). PIK3CD encodes PI3K-p110δ, which is the key component of PI3Kδ. Reintroduction of PIK3CD impaired WSX1-induced PD-L1 reduction in both SNU449 (n = 5 independent experiments, P = 0.0001) and SNU475 cells (P = 0.0013). g Effect of WSX1 on PIK3CD mRNA levels in SNU449 (n = 3 independent experiments P = 0.0139) and SNU475 HCC cells (P = 0.0006). h A proposed model illustrating that WSX1 facilitates antitumor immune surveillance through inhibiting the PI3Kδ/AKT/GSK3β/PD-L1 signaling pathway. Under physiological conditions, highly expressed WSX1 in hepatocytes transcriptionally downregulates PIK3δ, thereby reducing AKT activation and subsequently liberating GSK3β kinase activity from inhibition by AKT, leading to boosted GSK3β-mediated PD-L1 degradation. Without excessive PD-L1 expression on tumor cells, effector CD8⁺ T cells maximize their killing effect, resulting in inhibition of HCC development (Left). Otherwise, lack of WSX1 results in uncontrolled neoplastic PD-L1 expression and, ultimately, tumor immune evasion (Right). i Schematic diagram of the interactions among WSX1, PI3Kδ, AKT, GSK3β, and PD-L1. Quantitative data are presented as mean ± SD and were analyzed by one-way ANOVA or Student t test. Tukey-Kramer multiple comparison test was used for pairwise comparisons in the ANOVA analysis. Correlation analyses were performed by Pearson correlation test. Unless otherwise noted, data and images shown are representative of 3 independent experiments. All statistical tests were two-sided. *P < 0.05, **P < 0.01, ***P < 0.001. myr-AKT myristoylated AKT. The gating strategy for sorting PD-L1⁺ HCC cells is shown in supplementary Fig. 8b. Source data are provided as a Source Data file.