A three‑gene signature correlated with MAPK/ERK activation characterizes acquired resistance to EGFR‑tyrosine kinase inhibitors in non‑small cell lung cancer

Epidermal growth factor receptor‑targeted therapies such as afatinib provide clinical benefits to patients with advanced‑stage non‑small cell lung cancer (NSCLC); however, acquired resistance frequently develops, with the underlying mechanisms remaining undefined in 20‑30% of cases. The present study established afatinib‑resistant (AR) NSCLC cell lines and confirmed their resistance phenotype using Cell Counting Kit‑8 (CCK‑8) cell viability assays. Notably, these cells also exhibited cross‑resistance to osimertinib. To elucidate the molecular basis of resistance acquisition, the time‑resolved transcriptomic profiling of A549 cells was performed across three stages: Parental, afatinib‑exposed (adaptive phase) and stable resistant cells. The analyzed results revealed the persistent upregulation of ABLIM3, HTR1D and HSPA1A, which was validated by reverse transcription‑quantitative polymerase chain reaction. The meta‑analysis of hazard ratios from The Cancer Genome Atlas demonstrated that the elevated expression level of the three‑gene signature was significantly associated with tumor progression and an increased risk of disease recurrence. These transcriptional alterations were accompanied by the sustained activation of the MAPK/ERK signaling pathway, as evidenced by increased ERK1/2 phosphorylation detected using western blot analysis, which was positively associated with the expression level of the three‑gene signature. Functional analyses further demonstrated that the pharmacological inhibition of MAPK/ERK signaling using selumetinib effectively re‑sensitized AR cells to both afatinib and osimertinib, as demonstrated by restored drug sensitivity in CCK‑8 assays. Collectively, these findings suggest that MAPK/ERK signaling contributes to the transition from adaptive tolerance to stable resistance to afatinib and highlight a tractable therapeutic vulnerability for overcoming resistance to tyrosine kinase inhibitors in NSCLC.