uPAR通过MAPK信号抑制细胞自噬促进胰腺癌增殖、侵袭及化疗抵抗的作用研究

doi:10.19401/j.cnki.1007-3639.2024.10.004

摘要/Abstract

摘要：

背景与目的：尿激酶型纤溶酶原激活物受体（urokinase-type plasminogen activator receptor，uPAR）基因扩增与胰腺癌患者的不良预后密切相关。uPAR通过丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPK）信号转导通路调控胰腺癌细胞的上皮-间质转化（epithelial-mesenchymal transformation，EMT）和化疗抵抗，但具体机制尚未完全阐明。本研究旨在探讨uPAR通过抑制细胞自噬促进胰腺癌细胞增殖、侵袭和化疗抵抗的机制。方法：收集2021年12月—2022年6月在南华大学附属长沙中心医院（长沙市中心医院）接受手术切除和穿刺活检患者的胰腺癌组织标本[获得长沙市中心医院医院伦理委员会批准，编号：2021-S0182，2022-S0084]，体外培养胰腺癌患者来源类器官（patient-derived organoids，PDO）；使用6种胰腺癌细胞系（AsPC-1、PANC-1、CAPAN-1、CAPAN-2、MIA PaCa-2和PaTu8988T），利用成簇规律间隔短回文重复序列及其关联核酸酶9（clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9，CRISPR/Cas9）技术构建uPAR缺陷模型。采用共聚焦显微镜、蛋白质印迹法（Western blot）、酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）和MTS实验测定细胞增殖、侵袭能力，并分析MAPK和自噬信号通路以及吉西他滨诱导的细胞死亡变化。采用RNA干扰（siRNA）或自噬抑制剂评估联合治疗的协同作用。结果：在AsPC-1细胞中，uPAR敲除后，MTS实验和损伤修复实验结果显示，与野生型细胞相比，克隆细胞的增殖和迁移能力显著降低，对吉西他滨的敏感性降低。uPAR重新表达后，克隆细胞的增殖和侵袭能力恢复，且对吉西他滨的敏感性部分恢复。共聚焦显微镜结果显示，克隆细胞的F-肌动蛋白减少，细胞变得圆滑。Western blot结果显示，与野生型细胞相比，克隆细胞中E-钙黏蛋白（E-cadherin）和Slug表达升高，波形蛋白（vimentin）表达降低；磷酸化-黏着斑激酶（phospho-focal adhesion kinase，p-FAK）、p-p38MAPK和微管相关蛋白1的轻链3B（microtubule-associated protein light chain 3B，LC3B）表达升高（P<0.05）。siRNA结果表明，沉默FAK或p38MAPK或联合自噬抑制均能使克隆细胞对吉西他滨重新敏感，沉默p38MAPK能降低LC3B表达。类器官研究显示，8例类器官对吉西他滨的响应存在差异，uPAR在8例胰腺癌类器官中均有表达，uPAR表达水平与吉西他滨的半数抑制浓度（half-maximal inhibitory concentration，IC₅₀）呈负相关（r²=0.66，P<0.05），3例类器官对吉西他滨联合自噬抑制剂响应良好（P<0.05）。结论：uPAR通过抑制胰腺癌中依赖p38MAPK信号通路的细胞活性，防止FAK介导的耐药性和细胞休眠。本研究提示，uPAR高表达的胰腺癌患者对吉西他滨反应更好，而uPAR低表达且p38MAPK高表达的肿瘤可能从联合使用自噬抑制剂和细胞毒性化疗药物治疗中受益。

关键词: 胰腺癌, 类器官培养, uPAR, 自噬, 吉西他滨, 化疗抵抗

Abstract:

Background and purpose: Amplification of the urokinase plasminogen activator receptor (uPAR) gene is closely associated with poor prognosis in pancreatic cancer patients. uPAR regulates epithelial-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer cells through the mitogen-activated protein kinases (MAPK) signaling pathway, though the specific mechanisms remain unclear. This study aimed to investigate the mechanism by which uPAR promotes proliferation, invasion, and chemoresistance of pancreatic cancer cells by inhibiting autophagy. Methods: Pancreatic cancer tissue samples were collected from patients who underwent surgical resection and biopsy at the Changsha Central Hospital, Affiliated to University of South China (Changsha Central Hospital), between December 2021 and Jun 2022. The study was approved by the Ethics Committee of Changsha Central Hospital (Approval No.: 2021-S0182, 2022-S0084). Patient-derived organoids (PDOs) from pancreatic cancer samples were cultured in vitro. Six pancreatic cancer cell lines (AsPC-1, PANC-1, CAPAN-1, CAPAN-2, MIA PaCa-2 and PaTu8988T) were used in this study. uPAR-deficient models were constructed using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 technology. Cell proliferation and invasion abilities were measured using confocal microscopy, Western blot, enzyme-linked immunosorbent assay (ELISA), and MTS assays. Changes in MAPK and autophagy signaling pathways and gemcitabine-induced cell death were analyzed. The synergistic effects of combined treatments were evaluated using gene silencing (siRNA) or autophagy inhibitors. Results: In AsPC-1 cells, uPAR knockout significantly reduced the proliferation and migration abilities of clone cells compared to wild-type cells, as shown by MTS assays and wound healing experiments, and decreased sensitivity to gemcitabine (P<0.05). Re-expression of uPAR restored the proliferation and invasion abilities of clone cells and partially restored sensitivity to gemcitabine (P<0.05). Confocal microscopy revealed reduced F-actin and a rounded morphology in clone cells. Western blot analysis showed increased expressions of E-cadherin and Slug, decreased expression of vimentin, and increased expressions of phospho-focal adhesion kinase (p-FAK), p-p38MAPK, and the microtubule-associated protein light chain 3B (LC3B) in clone cells compared to wild-type cells. siRNA results indicated that silencing FAK or p38MAPK or combining autophagy inhibition could resensitize clone cells to gemcitabine (P<0.05), with p38MAPK silencing reducing LC3B expression. Organoid studies showed varying responses to gemcitabine among 8 organoids, all expressing uPAR. uPAR expression levels were negatively correlated with gemcitabine IC₅₀ (r²=0.66, P<0.05). Three organoids responded well to the combination of gemcitabine and autophagy inhibitors (P<0.05). Conclusion: uPAR promotes pancreatic cancer cell activity through the p38MAPK signaling pathway, preventing FAK-mediated resistance and cell dormancy. The study suggests that pancreatic cancer patients with high uPAR expression respond better to gemcitabine, while tumors with low uPAR and high p38MAPK expressions may benefit from combined treatment with autophagy inhibitors and cytotoxic chemotherapy.

Key words: Pancreatic cancer, Organoid, uPAR, Autophagy, Gemcitabine, Chemoresistance

中图分类号:

R735.9

谭小浪, 姚莎, 王桂华, 彭罗根. uPAR通过MAPK信号抑制细胞自噬促进胰腺癌增殖、侵袭及化疗抵抗的作用研究[J]. 中国癌症杂志, 2024, 34(10): 944-956.

TAN Xiaolang, YAO Sha, WANG Guihua, PENG Luogen. Research on uPAR promoting proliferation, migration, and chemoresistance of pancreatic cancer by inhibiting autophagy via MAPK signaling[J]. China Oncology, 2024, 34(10): 944-956.

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