Afatinib alters DNA methylation and Paneth-like differentiation markers in Caco-2 cells.

Afatinib is an irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) used to treat EGFR-mutant non-small cell lung cancer. It frequently causes gastrointestinal toxicity that perturbs intestinal homeostasis, and its impact on Paneth-like lineage differentiation along the crypt-villus axis remains unclear. Using a controlled differentiation-stage framework in Caco-2 cells, we examined how afatinib affects Paneth-like differentiation markers. We incubated undifferentiated Caco-2 cells with afatinib (10-5000 nM) for 24 h during early culture and evaluated downstream DNA methylation and differentiation-associated readouts up to day 14. We quantified proteins (western blotting), mRNAs (RT-qPCR), and promoter methylation (methylation-sensitive restriction enzyme-qPCR). Afatinib increased DNA methylation at the SRY-box transcription factor 9 (SOX9) and defensin alpha 5 (DEFA5) promoters and reduced their protein expression. Immunostaining indicated reduced expression of Paneth-like differentiation markers in Caco-2 cells. In contrast, mRNA levels of the SOX9 regulators odd-skipped related transcription factor 1 (OSR1) and receptor-interacting protein 140 (RIP140) were upregulated without changes in promoter methylation at the analyzed sites, indicating DNA methylation-independent regulation at these promoters. These findings suggest that DNMT1/3B-skewed methylation at the SOX9/DEFA5 promoters may be counteracted by ten-eleven translocation-mediated counter-demethylation. Collectively, our data indicate that afatinib modulates Paneth-like differentiation markers via DNA methylation-dependent repression of SOX9/DEFA5 and DNA methylation-independent induction of OSR1/RIP140 in Caco-2 cells, which may be relevant to crypt-associated epithelial function and gastrointestinal safety.