Comparison and Analysis of the Drug-Resistance Mechanism of Osimertinib- and Almonertinib-Resistant Cell Lines.

Abstract

Background: Non-small-cell lung cancer remains the leading cause of cancer-related deaths globally, and epidermal growth factor receptor mutations have been identified as crucial drivers of the disease. Encouragingly, epidermal growth factor receptor tyrosine kinase inhibitors have demonstrated promising clinical outcomes. Nonetheless, the emergence of resistance to third-generation EGFR-TKIs like osimertinib and almonertinib is an inevitable challenge. Methods: In this study, we generated almonertinib-resistant cell lines from H-1975 and HCC827 lung cancer cell lines. We utilized various assays, including cell proliferation assays, hematoxylin and eosin staining, and cell cycle assays, to investigate the characteristics of drug-resistant cells. Additionally, we performed RNA transcriptome sequencing to identify differentially expressed genes (DEGs) in almonertinib-resistant cells. To further expand our analysis, we obtained sequencing data of osimertinib-resistant cells from the Gene Expression Omnibus (GEO) dataset and identified DEGs in these cells. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to assess the biological functions and signaling mechanisms associated with DEGs. Furthermore, the survival prognosis and immune cell infiltration of common differentially expressed genes (co-DEGs) in osimertinib-and almonertinib-resistant cells were analyzed, and the expression of a co-DEG (IGFBP7) was verified through quantitative reverse transcriptase polymerase chain reaction (qPCR) and western blotting (WB) assays. Gene knockdown plasmids were constructed for cell transfection, and the invasive ability of resistant cells was assessed using a Transwell assay following the knockdown of IGFBP7. Results: Experimental cell counting kit-8 cytotoxicity studies revealed intriguing findings regarding drug resistance in lung cancer cells. Specifically, the IC₅₀ values and resistance factors of H-1975 and HCC827 cells were found to be 1.9 nM and 833.58 and 2.2 nM and 631.95, respectively. In addition to these quantitative results, comparative observations of the cell morphology and cell cycle revealed significant alterations in drug-resistant cells. Transcriptome sequencing analysis identified 220 DEGs between H-1975 and H-1975/AR and 736 DEGs between HCC827 and HCC827/AR. Interestingly, screening of overlapping DEGs with osimertinib-resistant cells in the GEO database identified some common genes, such as IGFBP7 and RFTN1, which were found to be associated with the improved prognosis of non-small-cell lung cancer by survival analysis. Furthermore, GO analysis and KEGG pathway enrichment analysis revealed different pathway changes in different drug-resistant cells. Survival analysis indicated that a higher expression of co-DEGs (IGFBP7, RFTN1) was associated with a more favorable prognosis. Furthermore, IGFBP7 expression is strongly associated with infiltration levels of CD8+ T cells, Tregs, and macrophage cells in lung adenocarcinoma. Molecular biology experiments confirmed that the mRNA and protein expression level of IGFBP7 were over-expressed in almonertinib-resistance cells. H-1975/AR cells were transfected with si-IGFBP7, and the results of transfection were verified at the mRNA and protein levels. After knocking down gene expression, the IC₅₀ of the cells was 0.3 ± 0.02 µM, which was significantly lower than that of untransfected cells. Additionally, the invasion of cells in the knockdown group was repressed. Conclusions: These findings indicated that almonertinib and osimertinib exhibited distinct resistance mechanisms in vitro, underscoring the need for tailored treatment approaches.