Huang Huichao, Huang Fangling, Liang Xujun, Fu Ying, Cheng Zhe, Huang Yan, Chen Zhuchu, Duan Yankun, Chen Yongheng
Department of Infectious Disease, XiangYa Hospital, Central South University, Changsha 410008, China.
Department of Oncology, NHC Key Laboratory of Cancer Proteomics & State Local Joint Engineering Laboratory for Anticancer Drugs, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, China.
Pharmaceuticals (Basel). 2022 Dec 27;16(1):37. doi: 10.3390/ph16010037.
Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC.
Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo.
In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis.
Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.
阿法替尼是一种第二代酪氨酸激酶抑制剂（TKI），在鼻咽癌（NPC）中发挥其放射增敏作用。然而，阿法替尼介导的放射敏感性的详细机制在NPC中仍不清楚。
进行定量磷酸化蛋白质组学和生物信息学分析以阐明全局磷酸化蛋白变化。通过蛋白质免疫印迹法评估CD44-Stat3轴的活性和上皮-间质转化（EMT）相关标志物。使用伤口愈合和Transwell实验来确定阿法替尼联合放疗处理后细胞迁移水平。通过CCK-8实验检测细胞增殖。应用IL-6的药理学激动剂激活Stat3。用阿法替尼、放疗或阿法替尼与放疗联合处理异种移植小鼠模型，以检测阿法替尼在体内的放射敏感性。
在本研究中，我们发现阿法替尼引发了NPC细胞中全局蛋白磷酸化改变。此外，生物信息学分析表明阿法替尼抑制CD44-Stat3信号传导及随后的EMT过程。而且，功能实验证明阿法替尼联合放疗处理在体外和体内均显著阻碍细胞活力、迁移、EMT过程及CD44-Stat3活性。此外，Stat3的药理学刺激挽救了阿法替尼在NPC细胞中诱导的放射敏感性和生物学功能。这表明阿法替尼通过阻断CD44-Stat3轴的活性逆转EMT过程。
总体而言，本研究确定了阿法替尼作为放射增敏剂的分子机制，从而为NPC放射增敏提供了一种潜在有用的联合治疗方法和药物靶点。我们的研究结果描述了阿法替尼在放射敏感性和癌症治疗方面的新功能。
