[PHPS1 enhances PD-L1 serine phosphorylation by regulating ROS/SHP-2/AMPK activity to promote apoptosis of oral squamous cell carcinoma cells]

Abstract

Objectives: To investigate the mechanism of PHPS1 for promoting apoptosis of oral squamous cell carcinoma cells and the role of AMPK in regulating tumor angiogenesis under hypoxic conditions.

Methods: Human oral squamous cell carcinoma Ca9-22 cells cultured in hypoxic conditions (1% O₂) were inoculated subcutaneously in 16 nude mice, which were divided into control group and PHPS1 group (n=8) for treatment with 10% DMSO and 10% PHPS1 respectively. Tumor growth in the mice was monitored till 14 days after the treatment, and the xenografts were examined pathologically using HE staining. In Ca9-22 cells cultured in 1% O₂, the effect of PHPS1, compound C (an AMPK inhibitor), and their combination on expressions of SHP-2, AMPK, HIF-1α, PD-L1, caspase-8, caspase-3 and BAX were evaluated using Western blotting.

Results: In the tumor-bearing nude mice, PHPS1 treatment significantly inhibited tumor growth and neovascularization. HE staining showed significantly reduced tumor angiogenesis in PHPS1-treated mice. In Ca9-22 cells in hypoxic cultures, PHPS1 treatment significantly decreased the expression levels of SHP-2, HIF-1α, PD-L1, ERK2, STAT3 and VEGF and increased the expression of AMPK. The inhibitory effects of PHPS1 on HIF-1α and PD-L1 were obviously attenuated by the addition of compound C. PHPS1 also enhanced the expressions of caspase-3, caspase-8 and Bax proteins and increased the phosphorylation levels of PD-L1 and S195 in Ca9-22 cells, and these effects were effectively attenuated by compound C.

Conclusions: PHPS1 can enhance PD-L1 serine phosphorylation by regulating SHP-2/AMPK activity to promote apoptosis of oral squamous cell carcinoma cells under hypoxic conditions.

Keywords: PD-L1; SHP-2; mitogen-activated protein kinase; oral squamous cell carcinoma.

图1

PHPS1对裸鼠肿瘤生长的影响 Fig.1 Effect of PHPS1 on tumor growth in nude mice. A: Comparison of tumor growth between the two groups. B: Comparison of tumor volume between the two groups (Mean±SD). **P<0.05.

图2

PHPS1对口腔鳞状细胞癌细胞生长影响 Fig.2 Effect of PHPS1 on angiogenesis in oral squamous cell carcinoma xenografts in nude mice in control and PHPS1 groups. A: HE staining of the tumor sections (Scale bar=100 μm). B: Number of neovessels in the two groups (Mean±SD). ***P<0.01.

图3

PHPS1对口腔鳞状细胞癌细胞血管新生能力的影响 Fig.3 Effect of PHPS1 on angiogenesis in oral squamous cell carcinoma cells. A: Angiogenesis of Ca9-22 cells in different groups assessed by tube formation assay (Scale bar=500 μm). B: Number of neovessels in each group (Mean±SD). **P<0.05.

图4

PHPS1对ROS通路相关蛋白的影响 Fig.4 Effect of PHPS1 on expressions of ROS pathway-related proteins in Ca9-22 cells. A: Western blotting of p-SHP-2, p-AMPK, HIF-1α, PD-L1, p-ERK1/2, p-STAT3, and VEGF protein expression in Ca9-22 cells in control and PHPS1 groups. B: Comparison of protein expression levels between control and PHPS1 groups (Mean±SD). **P<0.05.

图5

AMPK抑制剂对p-SHP-2、p-AMPK、HIF-1α、PD-L1蛋白表达情况的影响 Fig.5 Effect of AMPK inhibitor on expressions of p-SHP-2, p-AMPK, HIF-1α, and PD-L1 proteins. A: Western blotting of p-SHP-2, p-AMPK, HIF-1α, and PD-L1 protein expression in Ca9-22 cells in different groups. B: Comparison of the protein expression levels. **P<0.05.

图6

AMPK抑制剂对capase-3、caspase-8、PD-L1、BAX蛋白表达情况影响 Fig.6 Effect of AMPK inhibitor on expressions of caspase-3, caspase-8, PD-L1, and BAX proteins. A: Western blotting of caspase-3, caspase-8, PD-L1 S195 phosphorylation and Bax protein expressions in Ca9-22 cells in different groups. B: Comparison of the protein expression levels. **P<0.05.

图7

PHPS1靶向ROS/SHP-2/AMPK活性进而促进PD-L1丝氨酸磷酸化进而加速肿瘤凋亡的机制示意图 Fig.7 Schematic diagram illustrating the mechanism by which PHPS1 targets the ROS/SHP-2/AMPK pathway, thereby promoting serine phosphorylation of PD-L1 and accelerating tumor apoptosis.