Tang Qingshan, Dong Jiachen, Zhang Feng, Zhao Dan, Yang Qi, Wen Jiayu, Sun Yuhao, Wei Jifu, Liu Zhixian
Jiangsu Key Laboratory, Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.
Jiangsu Cancer Hospital, The Affiliated Cancer Hospital of Nanjing Medical University, Jiangsu Institute of Cancer Research, Nanjing, China.
Front Pharmacol. 2025 Feb 13;16:1489210. doi: 10.3389/fphar.2025.1489210. eCollection 2025.
The tyrosine receptor kinase inhibitor (TRKi) entrectinib is used to treat neurotrophic tyrosine receptor kinase (NTRK) fusion-positive solid tumors and ROS1-positive patients. Despite its impressive efficacy against cancer, the clinical application is still limited by the central nervous system (CNS)-related toxicities. However, the precise mechanism of such CNS-related toxicities remains elusive.
The effect of entrectinib-induced nerve cell damage was evaluated by the nerve cells (PC12, HT22 and SK-N-SH) based models. Various assays, including CCK-8, colony formation and EdU incorporation assays were utilized to estimate the cellular viability and proliferation ability. Cell apoptosis was measured by flow cytometry. Next, transcriptome sequencing technology was performed to identify differentially expressed genes (DEGs). Gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) analysis and gene set enrichment analysis (GSEA) were applied to predict the potential functions of DEGs. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting assays were performed to measure the expressions of thrombospondin-1 (THBS1), TGF-β1, PI3K, AKT and phosphorylated AKT (p-AKT) in the entrectinib-treated nerve cells. Additionally, we Preliminary observed and validated whether THBS1 overexpression could rescue nerve cell damage and the abnormalities in PI3K-AKT and TGF-β signaling pathways.
Entrectinib significantly inhibited the nerve cells proliferation and colony formation, and induced nerve cells apoptosis. Transcriptome sequencing analysis and qRT-PCR revealed that THBS1 was downregulated within entrectinib treatment. KEGG and GSEA analysis also suggested that entrectinib directly caused the abnormalities in proliferation-related signaling pathway like PI3K-AKT pathway, and apoptosis-related signaling pathway including TGF-β pathway. We further demonstrated that THBS1, TGF-β1, PI3K, AKT and p-AKT were downregulated by entrectinib. Meanwhile, pretreatment with THBS1 overexpression plasmids significantly rescued nerve cells (PC12, HT22 and SK-N-SH) from cell death and the abnormalities in PI3K-AKT and TGF-β signaling pathways.
These results identified a critical role of entrectinib in promoting nerve cell damage by downregulating the expression of THBS1 while also inhibiting PI3K-AKT and TGF-β signaling pathways. Our findings will provide potential therapeutic targets for CNS-related toxicities.
酪氨酸受体激酶抑制剂（TRKi）恩曲替尼用于治疗神经营养性酪氨酸受体激酶（NTRK）融合阳性实体瘤和ROS1阳性患者。尽管其抗癌疗效显著，但临床应用仍受中枢神经系统（CNS）相关毒性的限制。然而，这种CNS相关毒性的确切机制仍不清楚。
通过基于神经细胞（PC12、HT22和SK-N-SH）的模型评估恩曲替尼诱导的神经细胞损伤作用。采用包括CCK-8、集落形成和EdU掺入试验在内的各种试验来评估细胞活力和增殖能力。通过流式细胞术检测细胞凋亡。接下来，进行转录组测序技术以鉴定差异表达基因（DEG）。应用基因本体（GO）、京都基因与基因组百科全书（KEGG）分析和基因集富集分析（GSEA）来预测DEG的潜在功能。进行定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹试验以检测恩曲替尼处理的神经细胞中血小板反应蛋白-1（THBS1）、转化生长因子-β1（TGF-β1）、磷脂酰肌醇-3激酶（PI3K）、蛋白激酶B（AKT）和磷酸化AKT（p-AKT）的表达。此外，我们初步观察并验证了THBS1过表达是否可以挽救神经细胞损伤以及PI3K-AKT和TGF-β信号通路的异常。
恩曲替尼显著抑制神经细胞增殖和集落形成，并诱导神经细胞凋亡。转录组测序分析和qRT-PCR显示，在恩曲替尼处理下THBS1表达下调。KEGG和GSEA分析还表明，恩曲替尼直接导致增殖相关信号通路如PI3K-AKT通路以及凋亡相关信号通路包括TGF-β通路的异常。我们进一步证明恩曲替尼下调了THBS1、TGF-β1、PI3K、AKT和p-AKT的表达。同时，用THBS1过表达质粒预处理可显著挽救神经细胞（PC12、HT22和SK-N-SH）免于细胞死亡以及PI3K-AKT和TGF-β信号通路的异常。
这些结果确定了恩曲替尼在通过下调THBS1表达同时抑制PI3K-AKT和TGF-β信号通路来促进神经细胞损伤中的关键作用。我们的发现将为CNS相关毒性提供潜在的治疗靶点。
