Synergistic effects and molecular mechanisms of pirfenidone combined with EGFR-TKIs in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality globally. While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as osimertinib (OS) and afatinib (AF), have markedly improved clinical outcomes, their efficacy is significantly hindered by the development of acquired resistance. Emerging evidence suggests that p38γ is involved in the progression of NSCLC and the emergence of resistance to EGFR-TKIs. However, whether the p38γ inhibitor pirfenidone (PFD) can enhance the sensitivity of NSCLC to EGFR-TKIs remains unclear. This study aims to evaluate the synergistic antitumor effects of PFD in combination with OS or AF and to elucidate the underlying molecular mechanisms.

METHODS: EGFR-mutant NSCLC cell lines (PC9 and HCC827) and their OS-resistant derivatives (HCC827 OR) were utilized to evaluate cell proliferation by Cell Counting Kit-8 (CCK-8) assays and colony formation experiments. Western blotting was performed to analyze the expression of p38γ, proteins within the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway, and autophagy-related markers. Drug interactions were assessed using the SynergyFinder platform. Autophagic flux was investigated via a dual-fluorescent adenovirus (mRFP-GFP-LC3) and further validated using the autophagy inhibitor bafilomycin A1. Additionally, an HCC827 nude mouse xenograft model was employed for in vivo validation.

RESULTS: The combination of PFD with either OS or AF exhibited significant synergistic antitumor effects in both in vitro and in vivo NSCLC models, consistently achieving Bliss synergy scores exceeding 14. Notably, this combination successfully restored drug sensitivity in OS-resistant cells and reversed acquired resistance to EGFR-TKIs. Mechanistic investigations revealed that PFD enhanced EGFR-TKI sensitivity via two distinct pathways: firstly, by specifically inhibiting p38γ, a critical mediator of resistance, which leads to the downregulation of the PI3K/AKT/mTOR signaling pathway; and secondly, by promoting the autophagy cascade in a p38γ-independent manner. These synergistic effects were not reversed by ferroptosis or pyroptosis inhibitors and were only partially attributed to apoptosis.

CONCLUSIONS: PFD enhances the efficacy of EGFR-TKIs through a dual mechanism: first, by specifically inhibiting the p38γ-mediated PI3K/AKT/mTOR signaling pathway, and second, by activating a p38γ-independent autophagy pathway. The activation of the p38γ-mediated PI3K/AKT/mTOR pathway may represent a key mechanism underlying OS resistance. This study provides the first evidence that PFD can restore drug sensitivity in OS-resistant cells, offering valuable experimental support for its potential clinical application as an EGFR-TKI sensitizer and a therapeutic strategy to overcome resistance in targeted cancer therapies.