Yan Rui, Huang Xuying, Liu Heshu, Xiao Zeru, Liu Jian, An Guangyu, Ge Yang
Beijing Chao-Yang Hospital Department of Oncology, Capital Medical University, 8 Gongren Tiyuchang Nanlu Road, Chaoyang Dist., Beijing 100020, China.
Biomedicines. 2023 May 22;11(5):1490. doi: 10.3390/biomedicines11051490.
Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a first-line treatment for lung adenocarcinoma with EGFR-sensitive mutations, but acquired resistance to EGFR-TKIs remains a problem in clinical practice. The development of epithelial-mesenchymal transition (EMT) is a critical mechanism that induces acquired resistance to TKIs. Reversing acquired resistance to EGFR-TKIs through targeting the key molecules driving EMT provides an alternative choice for patients. We, therefore, aimed to explore the role of doublecortin-like kinase 1 (DCLK1) as an EMT driver gene in the acquired resistance of lung adenocarcinoma to EGFR-TKIs.
The IC of Gefitinib or Osimertinib in PC9/HCC827 cells was measured using a cell counting kit-8 (CCK8) assay. The expression levels of EMT-related genes in PC9 and HCC827 cells were detected using RT-PCR and Western blot. Cell migration and invasion abilities were assessed via a transwell assay. For the in vivo experiments, PC9 cells were subcutaneously injected into BALB/c nude mice to form tumors. Upon harvesting, tumor tissues were retained for RT-PCR, Western blot, and polychromatic fluorescence staining to detect biomarker changes in the EMT process.
Gefitinib-resistant PC9 (PC9/GR) and Osimertinib-resistant HCC827 (HCC827/OR) cells showed remarkable activation of EMT and enhanced migration and invasion abilities compared to TKI-sensitive cells. In addition, DCLK1 expression was markedly increased in EGFR-TKI-resistant lung adenocarcinoma cells. The targeted knockout of DCLK1 effectively reversed the EMT phenotype in TKI-resistant cells and improved EGFR-TKI sensitivity, which was further validated by the in vivo experiments.
DCLK1 facilitates acquired resistance to EGFR-TKI in lung adenocarcinoma by inducting EMT and accelerating the migration and invasion abilities of TKI-resistant cells.
表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是表皮生长因子受体（EGFR）敏感突变型肺腺癌的一线治疗药物，但在临床实践中，对EGFR-TKI获得性耐药仍是一个问题。上皮-间质转化（EMT）的发生是诱导对酪氨酸激酶抑制剂（TKIs）获得性耐药的关键机制。通过靶向驱动EMT的关键分子来逆转对EGFR-TKIs的获得性耐药为患者提供了一种替代选择。因此，我们旨在探讨双皮质素样激酶1（DCLK1）作为EMT驱动基因在肺腺癌对EGFR-TKIs获得性耐药中的作用。
使用细胞计数试剂盒-8（CCK8）检测吉非替尼或奥希替尼在PC9/HCC827细胞中的半数抑制浓度（IC）。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测PC9和HCC827细胞中EMT相关基因的表达水平。通过Transwell实验评估细胞迁移和侵袭能力。在体内实验中，将PC9细胞皮下注射到BALB/c裸鼠体内形成肿瘤。收获后，保留肿瘤组织进行RT-PCR、蛋白质免疫印迹法和多色荧光染色，以检测EMT过程中的生物标志物变化。
与TKI敏感细胞相比，吉非替尼耐药的PC9（PC9/GR）细胞和奥希替尼耐药的HCC827（HCC827/OR）细胞显示出明显的EMT激活以及增强的迁移和侵袭能力。此外，在EGFR-TKI耐药的肺腺癌细胞中，DCLK1表达显著增加。靶向敲除DCLK1有效地逆转了TKI耐药细胞中的EMT表型并提高了EGFR-TKI敏感性，体内实验进一步验证了这一点。
DCLK1通过诱导EMT和加速TKI耐药细胞的迁移和侵袭能力，促进肺腺癌对EGFR-TKI的获得性耐药。
