Unmasking the common enemy: drug resistance mechanisms across three different EGFR inhibitor generations are associated with co-targetable alterations in extracellular matrix signaling.

backgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have transformed non-small cell lung cancer (NSCLC) treatment, offering substantial survival benefits. However, acquired resistance remains a significant obstacle, undermining long-term efficacy. While specific mechanisms of EGFR-TKI resistance have been reported, potential shared mechanisms across EGFR-TKI generations have remained unclear.

methodsDrug-resistant HCC827 and NCI-H1975 cells were developed by a 10-month stepwise selection using gefitinib, dacomitinib, and osimertinib, representing first, second, and third EGFR-TKI generations, respectively. Global proteomes of the resistant and parental NSCLC cell lines were compared using liquid chromatography tandem mass spectrometry. After bioinformatic pathway analyses, the candidate protein and gene alterations were validated by western blotting and droplet digital PCR. Drug (cross-)resistance patterns and reversal responses were quantified by MTT assay after single and combination drug treatments, or gene silencing by small interfering RNA. Invasive cell activities were evaluated in spheroid models within three-dimensional (3D) collagen matrices. Bioinformatics analyses of open-access transcriptomic datasets were applied to explore gene expression alterations linked to clinical EGFR mutations and disease prognosis and relapse.

resultsWe established EGFR-TKI-resistant NSCLC cell lines for all three drug generations, demonstrating cross-resistance and significantly enhanced invasive potential in 3D collagen. Notably, extracellular matrix components and signaling proteins (FN1, FAK, YAP1) were found altered and validated as synergistically targetable resistance drivers across all three drug generations, irrespective of cellular background. Moreover, the second- and third-generation models shared co-targetable dysregulations in cancer stemness regulators (Hedgehog, Notch), anti-apoptotic protein BCL-2, and the drug efflux transporter ABCG2. Finally, transcriptomic analysis showed that in human EGFR-positive NSCLC tumors, overexpression of FN1 and collagen-related genes was associated with post-treatment relapse and poor patient survival.

conclusionsThis study identifies/validates key mechanisms of EGFR-TKI resistance and suggests combinatorial therapeutic strategies as potential interventions against EGFR-TKI-resistant lung cancers, with promising implications for improving clinical outcomes.