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

doi:10.12122/j.issn.1673-4254.2024.12.24

Abstract

Abstract:

Objective 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. Conclusion 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.

Key words: SHP-2, mitogen-activated protein kinase, oral squamous cell carcinoma, PD-L1

Jinhong ZHANG, Xin LIU, Jian LIU. PHPS1 enhances PD-L1 serine phosphorylation by regulating ROS/SHP-2/AMPK activity to promote apoptosis of oral squamous cell carcinoma cells[J]. Journal of Southern Medical University, 2024, 44(12): 2469-2476.

Figures/Tables 7

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.

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.

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.

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.

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.

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.

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.

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