Yang Li-Yan, Cheng Zhi-Jian, Liu Zou, Wang Di, Zhang Na, Fan Zhi-Lu, Cai Hong-Qing, Zhang Yu, Cai Yan, Xu Xin, Wang Jin-Hua, Du Guan-Hua, Hao Jia-Jie, Wang Ming-Rong
State Key Laboratory of Molecular Oncology, Center for Cancer Precision Medicine, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Department of Neurosurgery, The People's Hospital of Huangshan, Huangshan, China.
J Gastroenterol Hepatol. 2021 Sep;36(9):2513-2522. doi: 10.1111/jgh.15490. Epub 2021 Apr 1.
Chemotherapy drugs do not work well in esophageal squamous cell carcinoma (ESCC), and none of the targeted drugs have been applied in clinic. This study aims to identify effective targeted drugs and related biomarkers for the treatment of ESCC.
The effect of 40 Food and Drug Administration-approved small-molecule inhibitors was first tested in five ESCC cell lines. CCK8 assays and xenografts derived from ESCC cell lines were performed to evaluate the anti-ESCC effects of inhibitors or chemotherapeutic agents in vitro and in vivo, respectively. Immunohistochemistry was utilized to analyze the p-EGFR expression in tissues. Western blot combining with gray analysis was conducted to detect the expression of interest protein. Flow cytometry and immunofluorescence assay were used to analyze apoptosis, cell cycle, and mitotic changes after drug treatment.
Afatinib showed remarkable effects on inhibiting ESCC cells with higher expression of p-EGFR. Results from combinatorial screening in ESCC cells expressing lower phosphorylation level of EGFR showed that paclitaxel and afatinib presented a significant synergistic inhibitory effect (P < 0.001). Molecular analysis revealed that paclitaxel sensitized afatinib by activating EGFR, and afatinib in combination with paclitaxel effectively blocked MAPK pathway and induced G2/M cell arrest and apoptosis that is an indicator of mitotic catastrophe.
Our data demonstrate that afatinib is an effective drug for patients with ESCC expressing higher phosphorylation level of EGFR. And for patients with lower p-EGFR in tumors, paclitaxel in combination with afatinib might be a promising therapeutic strategy in ESCC.
化疗药物对食管鳞状细胞癌（ESCC）疗效不佳，且尚无靶向药物应用于临床。本研究旨在确定治疗ESCC的有效靶向药物及相关生物标志物。
首先在5种ESCC细胞系中测试40种美国食品药品监督管理局批准的小分子抑制剂的效果。分别进行CCK8检测和源自ESCC细胞系的异种移植，以评估抑制剂或化疗药物在体外和体内的抗ESCC作用。利用免疫组织化学分析组织中p-EGFR的表达。进行蛋白质免疫印迹结合灰度分析以检测目的蛋白的表达。采用流式细胞术和免疫荧光分析药物处理后细胞的凋亡、细胞周期及有丝分裂变化。
阿法替尼对抑制p-EGFR表达较高的ESCC细胞显示出显著效果。在EGFR磷酸化水平较低的ESCC细胞中的联合筛选结果表明，紫杉醇和阿法替尼呈现出显著的协同抑制作用（P < 0.001）。分子分析显示，紫杉醇通过激活EGFR使阿法替尼敏感，阿法替尼与紫杉醇联合可有效阻断MAPK通路并诱导G2/M期细胞阻滞和凋亡，这是有丝分裂灾难的一个指标。
我们的数据表明，阿法替尼对EGFR磷酸化水平较高的ESCC患者是一种有效药物。对于肿瘤中p-EGFR较低的患者，紫杉醇联合阿法替尼可能是ESCC中一种有前景的治疗策略。
