PHPS1通过调控口腔鳞状细胞癌内ROS/SHP-2/AMPK活性促进PD-L1丝氨酸磷酸化进而加速肿瘤凋亡

doi:10.12122/j.issn.1673-4254.2024.12.24

摘要/Abstract

摘要：

目的探讨PHPS1通过对口腔鳞状细胞癌细胞ROS/酪氨酸磷酸酶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研究，分析腺苷酸活化蛋白激酶（AMPK）对于低氧环境下肿瘤内血管生成的影响。方法 6~8周龄健康裸鼠16只，皮下移植瘤模型分为Control组和PHPS1组， 8只/组，培育14 d后观察肿瘤的生长情况，并将其切片进行HE染色固定并拍照。将人口腔鳞状细胞癌Ca9-22细胞系培养后分为Control组、PHPS1组、Control+Compound C组（Compound C为AMPK抑制剂）、PHPS1+Compound C组在1%低氧环境下培养，通过Western blotting对细胞内SHP-2、AMPK、HIF-1α、PD-L1、caspase-8、caspase-3和BAX含量进行检测。结果裸鼠成瘤与血管新生实验结果显示PHPS1抑制了裸鼠体内肿瘤生长和血管新生（P<0.05）。Western blotting分析显示PHPS1降低了SHP-2、HIF-1α、PD-L1、ERK2、STAT3和VEGF的表达，同时增加了AMPK的表达（P<0.05）。加入AMPK抑制剂后，PHPS1对HIF-1α和PD-L1的抑制作用减弱（P<0.05）。此外，PHPS1促进了caspase-3、caspase-8、PD-L1 S195磷酸化和Bax蛋白的表达，这些效应在加入AMPK抑制剂后也有所减弱（P<0.05）。HE染色结果表明PHPS1组肿瘤血管生成数量减少（P<0.01）。结论在缺氧环境下可以通过调节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。

关键词: SHP-2, AMPK, 口腔鳞状细胞癌, PD-L1

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

张晋弘, 刘昕, 刘健. PHPS1通过调控口腔鳞状细胞癌内ROS/SHP-2/AMPK活性促进PD-L1丝氨酸磷酸化进而加速肿瘤凋亡[J]. 南方医科大学学报, 2024, 44(12): 2469-2476.

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.

图/表 7

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.

参考文献 30

1	Renu K, Vinayagam S, Veeraraghavan VP, et al. Molecular crosstalk between the immunological mechanism of the tumor microenvironment and epithelial-mesenchymal transition in oral cancer[J]. Vaccines, 2022, 10(9): 1490.
2	Grégoire V, Grau C, Lapeyre M, et al. Target volume selection and delineation (T and N) for primary radiation treatment of oral cavity, oropharyngeal, hypopharyngeal and laryngeal squamous cell carcinoma[J]. Oral Oncol, 2018, 87: 131-7.
3	Porceddu SV, Daniels C, Yom SS, et al. Head and neck cancer international group (HNCIG) consensus guidelines for the delivery of postoperative radiation therapy in complex cutaneous squamous cell carcinoma of the head and neck (cSCCHN)[J]. Int J Radiat Oncol Biol Phys, 2020, 107(4): 641-51.
4	Woo SB. Oral epithelial dysplasia and premalignancy[J]. Head Neck Pathol, 2019, 13(3): 423-39.
5	Bai YC, Boath J, White GR, et al. The balance between differentiation and terminal differentiation maintains oral epithelial homeostasis[J]. Cancers, 2021, 13(20): 5123.
6	Niogret C, Birchmeier W, Guarda G. SHP-2 in lymphocytes' cytokine and inhibitory receptor signaling[J]. Front Immunol, 2019, 10: 2468.
7	Wu XL, Guan SY, Lu YG, et al. Macrophage-derived SHP-2 inhibits the metastasis of colorectal cancer via Tie2-PI3K signals[J]. Oncol Res, 2023, 31(2): 125-39.
8	Feng GS. Shp2-mediated molecular signaling in control of embryonic stem cell self-renewal and differentiation[J]. Cell Res, 2007, 17(1): 37-41.
9	Spaulding HR, Yan Z. AMPK and the adaptation to exercise[J]. Annu Rev Physiol, 2022, 84: 209-27.
10	Ciccarese F, Zulato E, Indraccolo S. LKB1/AMPK pathway and drug response in cancer: a therapeutic perspective[J]. Oxid Med Cell Longev, 2019, 2019: 8730816.
11	Hsu CC, Peng DN, Cai Z, et al. AMPK signaling and its targeting in cancer progression and treatment[J]. Semin Cancer Biol, 2022, 85: 52-68.
12	Karampitsakos T, Galaris A, Barbayianni I, et al. SH2 domain-containing phosphatase-SHP2 attenuates fibrotic responses through negative regulation of mitochondrial metabolism in lung fibroblasts[J]. Diagnostics, 2023, 13(6): 1166.
13	Jiang JH, Hu BJ, Chung CS, et al. SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a combined murine hemorrhage followed by septic challenge model[J]. Mol Med, 2020, 26(1): 89.
14	Salem IH, Plante S, Gounni AS, et al. A shift in the IL-6/STAT3 signalling pathway imbalance towards the SHP2 pathway in severe asthma results in reduced proliferation process[J]. Cell Signal, 2018, 43: 47-54.
15	Tan SZ, Li DP, Zhu X. Cancer immunotherapy: pros, cons and beyond[J]. Biomed Pharmacother, 2020, 124: 109821.
16	Sobierajska K, Ciszewski WM, Sacewicz-Hofman I, et al. Endothelial cells in the tumor microenvironment[J]. Adv Exp Med Biol, 2020, 1234: 71-86.
17	Xu ZY, Guo CY, Ye QL, et al. Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization[J]. Nat Commun, 2021, 12(1): 6310.
18	Chen H, Cresswell GM, Libring S, et al. Tumor cell-autonomous SHP2 contributes to immune suppression in metastatic breast cancer[J]. Cancer Res Commun, 2022, 2(10): 1104-18.
19	Zhao H, Ren XJ, Kong RY, et al. Auxilin regulates intestinal stem cell proliferation through EGFR[J]. Stem Cell Reports, 2022, 17(5): 1120-37.
20	Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis[J]. Nat Rev Mol Cell Biol, 2018, 19(2): 121-35.
21	Chun Y, Kim J. AMPK-mTOR signaling and cellular adaptations in hypoxia[J]. Int J Mol Sci, 2021, 22(18): 9765.
22	Cha JH, Yang WH, Xia WY, et al. Metformin promotes antitumor immunity via endoplasmic-reticulum-associated degradation of PD-L1[J]. Mol Cell, 2018, 71(4): 606-20. e7.
23	Cha JH, Chan LC, Li CW, et al. Mechanisms controlling PD-L1 expression in cancer[J]. Mol Cell, 2019, 76(3): 359-70.
24	Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point[J]. Nature, 2017, 541(7637): 321-30.
25	Yao H, Lan J, Li CS, et al. Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours[J]. Nat Biomed Eng, 2019, 3(4): 306-17.
26	Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation[J]. Cell, 2011, 144(5): 646-74.
27	Mabeta P, Steenkamp V. The VEGF/VEGFR axis revisited: implications for cancer therapy[J]. Int J Mol Sci, 2022, 23(24): 15585.
28	Wang ZQ, Zhao P, Tian KH, et al. TMEM9 promotes lung adenocarcinoma progression via activating the MEK/ERK/STAT3 pathway to induce VEGF expression[J]. Cell Death Dis, 2024, 15(4): 295.
29	Lee SH, Golinska M, Griffiths JR. HIF-1-independent mechanisms regulating metabolic adaptation in hypoxic cancer cells[J]. Cells, 2021, 10(9): 2371.
30	Ma S, Lu CC, Yang LY, et al. ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis[J]. J Exp Clin Cancer Res, 2018, 37(1): 183.

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