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Updated: Jul 17, 2025
Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib
Published on: June 26, 2019
Characterization of circRNAs in established osimertinib‑resistant non‑small cell lung cancer cell lines
Xin Chen1, Jingyao Gu1, Jiali Huang2
1Cancer Medical Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210011, P.R. China.
Abstract:
Drug resistance is an urgent problem to be solved in the treatment of non‑small‑cell lung cancer (NSCLC). Osimertinib is a third‑generation EGFR‑tyrosine kinase inhibitor, which can improve the efficacy and quality of life of patients; however, the inevitable resistance after long‑term use of osimertinib often leads to treatment failure. Cell lines are key tools for basic and preclinical studies. At present, few osimertinib‑resistant cell lines (HCC827‑OR and H1975‑OR) have been established. In the present study, osimertinib‑resistant cell lines were established by gradually increasing the drug concentration. Half‑maximal inhibitory concentration (IC50), cell morphology, whole exon sequencing, Cell Counting Kit‑8 assay, EdU staining and flow cytometry were used to evaluate the osimertinib‑resistant cell lines. Western blot analysis was used to detect the expression levels of key proteins involved in osimertinib resistance. The circular RNA (circRNA) expression profile was identified by RNA sequencing (RNA‑seq) analysis of HCC827, HCC827‑OR, H1975 and H1975‑OR cells. Subsequently, the biological roles of differentially expressed circRNAs were explored in in vitro studies. Osimertinib‑resistant cell lines were successfully established via treatment with an increasing concentration of osimertinib. Osimertinib IC50 and proliferation of resistant cells were much higher than those of sensitive cells. Notably, phosphorylated (p)‑AKT and p‑ERK were markedly activated in resistant cells, and the inhibitory effect of osimertinib on p‑AKT and p‑ERK was weaker in resistant cells than that in parental cells. RNA‑seq analysis identified differentially expressed circRNAs in HCC827, HCC827‑OR, H1975 and H1975‑OR cells. The most dysregulated circRNAs (circPDLIM5 and circPPP4R1) were selected for further functional study. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the host genes of differentially expressed circRNAs were associated with 'endocytosis' and 'regulation of autophagy'. In conclusion, the present study established osimertinib‑resistant cell lines and revealed that circRNAs may serve as a promising biomarker in NSCLC osimertinib resistance.
Insights
Drug resistance to osimertinib is a major challenge in non-small cell lung cancer (NSCLC) treatment. This study established resistant cell lines and identified circular RNAs (circRNAs) as potential biomarkers for overcoming osimertinib resistance.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Drug resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor, is a significant obstacle in treating non-small cell lung cancer (NSCLC).
- Limited availability of osimertinib-resistant cell lines hinders preclinical research into resistance mechanisms.
- Understanding these mechanisms is crucial for developing strategies to improve patient outcomes and treatment efficacy.
Purpose of the Study:
- To establish and characterize osimertinib-resistant NSCLC cell lines.
- To investigate the molecular mechanisms underlying osimertinib resistance, focusing on protein expression and circular RNA profiles.
- To explore the potential of circular RNAs as biomarkers for osimertinib resistance in NSCLC.
Main Methods:
- Establishment of osimertinib-resistant cell lines (HCC827-OR, H1975-OR) through gradual drug concentration increase.
- Evaluation of resistance using IC50, cell morphology, whole exon sequencing, CCK-8 assay, EdU staining, and flow cytometry.
- Western blot analysis for key protein expression (p-AKT, p-ERK) and RNA sequencing (RNA-seq) for circRNA profiling.
Main Results:
- Successfully established osimertinib-resistant NSCLC cell lines with significantly higher IC50 and proliferation rates compared to sensitive cells.
- Activated p-AKT and p-ERK signaling pathways were observed in resistant cells, with diminished sensitivity to osimertinib's inhibitory effects.
- RNA-seq identified differentially expressed circRNAs, with circPDLIM5 and circPPP4R1 highlighted, and pathway analysis linked host genes to 'endocytosis' and 'regulation of autophagy'.
Conclusions:
- Osimertinib-resistant NSCLC cell lines were successfully generated, providing valuable tools for resistance research.
- Dysregulated circRNAs, particularly circPDLIM5 and circPPP4R1, are implicated in NSCLC osimertinib resistance.
- Circular RNAs show promise as potential biomarkers for predicting and potentially overcoming osimertinib resistance in NSCLC patients.
