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

Abstract

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.