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

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with a 5-year overall survival rate of only approximately 21% (1,2). According to global cancer statistics from 2024, lung cancer represents 12.8% of all tumor diagnoses and is responsible for 1.86 million deaths, comprising 19.1% of global cancer-related mortality (3). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers, with lung adenocarcinoma (LUAD) being the predominant histological subtype (4-6). Despite advances in early detection and systemic therapies, poor prognosis remains largely attributable to advanced-stage diagnosis, metastatic progression, and the development of therapeutic resistance.

Epidermal growth factor receptor (EGFR) is one of the most extensively characterized oncogenic drivers in lung cancer and remains, with activating mutations occurring in approximately 45% to 50% of Asian patients and 15% to 20% in Western populations (7). Exon 19 deletions and exon 21 L858R substitutions account for the majority of sensitizing EGFR alterations (8). EGFR tyrosine kinase inhibitors (TKIs), including the second-generation inhibitor afatinib (AF) and the third-generation inhibitor osimertinib (OS), have substantially improved outcomes for patients with EGFR-mutant NSCLC. OS, which effectively targets both sensitizing EGFR mutations and the T790M resistance mutation, has become a standard first-line treatment for advanced EGFR-mutant NSCLC (9,10). However, most patients ultimately develop acquired resistance within 1–2 years of treatment, resulting in disease progression and limiting the durability of clinical benefit (11,12). EGFR-TKI resistance can arise through EGFR-dependent mechanisms, such as secondary or tertiary EGFR alterations and enhanced EGFR signaling, or through EGFR-independent mechanisms involving bypass signaling, histological transformation, and tumor microenvironmental remodeling (13). Although multiple resistance mechanisms have been identified, effective therapeutic strategies to prevent or reverse EGFR-TKI resistance remain limited. Therefore, identifying novel agents that enhance EGFR-TKI sensitivity represents an important unmet clinical need.

Pirfenidone (PFD), a pyridinone derivative approved by the US Food and Drug Administration (FDA) for idiopathic pulmonary fibrosis (IPF), has attracted increasing attention due to its anti-inflammatory, anti-fibrotic, and potential anti-tumor properties. Although the precise molecular mechanisms underlying its effects remain incompletely understood, PFD has been reported to regulate fibrotic signaling pathways, suppress fibroblast activation, and modulate pathological tissue remodeling (14,15). Beyond fibrosis, accumulating evidence suggests that PFD exhibits antitumor activity through inhibition of tumor cell proliferation, regulation of cancer-associated fibroblast functions, and modulation of oncogenic signaling pathways in various cancer types (16-19). Importantly, recent studies have identified p38γ as a potential molecular target of PFD. p38γ, a member of the p38 mitogen-activated protein kinase (MAPK) family, participates in tumor-promoting processes including cell proliferation, survival, and therapy resistance (20,21). PFD-mediated inhibition of p38γ has been shown to suppress tumor progression and restore sensitivity to EGFR-TKI treatment in KRAS-mutant cancer models, suggesting its potential application in overcoming targeted therapy resistance (22,23).

Based on the clinical challenge of acquired EGFR-TKI resistance and the emerging antitumor and TKI-sensitizing properties of PFD, we hypothesized that PFD may enhance the therapeutic efficacy of EGFR-TKIs and delay or overcome acquired resistance in NSCLC. In this study, we investigated the antitumor activity and potential synergistic effects of PFD in combination with OS or AF in NSCLC models and explored the underlying molecular mechanisms. Our findings provide a potential rationale for repurposing PFD as an adjunctive strategy to enhance EGFR-TKI efficacy and overcome therapeutic resistance in EGFR-mutant NSCLC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0900/rc).

Methods

Clinical samples

Study design and patient screening

This study was conducted as a single-center retrospective observational study. Patients with EGFR-mutant NSCLC who received treatment at West China Hospital, Sichuan University from January 2019 to December 2024 were enrolled as study subjects. All included patients received OS monotherapy as first-line targeted therapy. Eligible cases were strictly screened according to the pre-specified inclusion and exclusion criteria, and a total of 201 valid cases were finally included in this study.

Inclusion criteria: (I) patients diagnosed with NSCLC confirmed by histopathological or cytological examination; (II) patients with confirmed EGFR gene mutations detected by molecular assays such as amplification refractory mutation system (ARMS) or next-generation sequencing (NGS), including exon 19 deletion, exon 21 L858R point mutation and other rare EGFR mutation subtypes; (III) patients received OS monotherapy as first-line treatment, with standardized medication and regular follow-up in our hospital; (IV) patients with complete baseline clinical data, gene testing results, laboratory examinations and imaging follow-up records, which could support efficacy and prognosis analysis; (V) patients had at least one measurable lesion at baseline defined by the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1).

Exclusion criteria: (I) patients complicated with other primary malignant tumors at baseline diagnosis; (II) patients who received other EGFR-TKIs, systemic chemotherapy, immunotherapy or other anti-tumor systemic therapy prior to OS treatment; (III) patients with severely missing clinicopathological information, gene testing results or follow-up data, which made efficacy and prognosis evaluation infeasible; (IV) patients who received combined systemic anti-tumor therapy (including chemotherapy, anti-angiogenic agents, immune checkpoint inhibitors, etc.) during OS treatment; (V) patients who were lost to follow-up throughout the follow-up period, with undetermined disease progression status and survival outcomes.

Ethical review

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was reviewed and approved by the Biomedical Ethics Review Committee of West China Hospital, Sichuan University [approval No. 2025 Annual Review (2716)] via expedited review. Given the retrospective nature of this study, all clinical data of enrolled patients were anonymized, and no interference with routine clinical diagnosis and treatment was involved during the study. Therefore, the ethics committee granted a waiver of informed consent for this study.

Clinical data collection

Complete clinical data of all enrolled patients were retrospectively collected from the hospital electronic medical record system, mainly including: (I) baseline demographic data: gender, age; (II) clinicopathological data: tumor stage, pathological type; (III) molecular testing data: EGFR mutation subtype; (IV) follow-up efficacy data: disease progression status, progression-free survival (PFS).

Statistical methods

Statistical software was used for data sorting and descriptive analysis of clinical samples. Categorical variables were presented as number of cases (n) and percentage (%). Continuous variables were described according to data distribution characteristics: normally distributed data were presented as mean, while skewed distributed data were presented as median.

Chemicals

OS (MCE, HY-15787), PFD (MCE, HY-B0673), AF (MCE, HY-10261), A83-01 (MCE, HY-10432), rapamycin (MCE, HY-10219), bafilomycin A1 (MCE, HY-100558), torin 2 (MCE, HY-13002), temsirolimus (MCE, HY-50910), SP600125 (MCE, HY-12041), ferrostatin-1 (MCE, HY-100579), birinapant (MCE, HY-16591), and belnacasan (MCE, HY-13205).

Antibodies

Primary antibodies against MAPK12/p38γ (Proteintech, 55132-1-AP), mTOR (Cell Signaling Technology, 2983), phospho-mTOR (Proteintech, 67778-1-Ig), PI3K (Cell Signaling Technology, 4257), phospho-PI3K (Cell Signaling Technology, 4228), AKT (Cell Signaling Technology, 4691), phospho-AKT (Ser473) (Cell Signaling Technology, 4060), SQSTM1/p62 (Cell Signaling Technology, 88588), Beclin-1 (Proteintech, 11306-1-AP), ATG5 (HUABIO, ET1607-5), LC3B (Abcam, ab192890), and GAPDH (Proteintech, 60004-1-Ig) were used. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Bioss, bs-0295G-HRP) served as the secondary antibody.

Cell culture

Human lung cancer cell lines HCC827, PC9, and H1975 were purchased from HySigen Biosciences Inc and authenticated by short tandem repeat (STR) profiling. Cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Biochannel) and maintained in a humidified incubator at 37 ℃ with 5% CO₂. All cell lines were routinely tested for mycoplasma contamination and confirmed to be negative.

Cell viability assay [Cell Counting Kit-8 (CCK-8 assay)]

Cells were seeded into 96-well plates and incubated overnight to allow adherence. Following attachment, cells were treated with the indicated compounds. At the designated time points, the culture medium was removed, and 100 μL of CCK-8 working solution (Vazyme, C6005), diluted 1:10 in serum-free RPMI-1640 medium, was added to each well. After incubation for 1 h at 37 ℃, absorbance was measured at 450 nm using a microplate reader. Cell viability or inhibition rate was calculated relative to the control group.

Colony formation assay

Cells were seeded at low density into 6-well plates and incubated overnight. After cell attachment, the medium was replaced with complete medium containing the indicated drugs. Half-medium changes were performed every 2–3 days with fresh drug-containing medium. Cells were cultured for 7–14 days until visible colonies formed. The medium was then removed, and cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.4% crystal violet for 15 min. After washing with PBS and air-drying, images were captured using a scanner or camera. Colony formation area was quantified using ImageJ software.

Drug interaction analysis using SynergyFinder

Following the CCK-8 assay, cell viability data under different drug combination concentrations were entered into an Excel file according to the template provided by the SynergyFinder web platform (https://synergyfinder.fimm.fi/). The data file was uploaded, and interaction effects were evaluated using established reference models, including the Highest Single Agent (HSA), Bliss independence, Loewe additivity, and zero interaction potency (ZIP) models. Analysis reports were subsequently generated and downloaded. The synergy score was used to quantify drug interactions: a score <−10 indicates potential antagonism, a score between −10 and 10 suggests an additive effect, and a score >10 indicates potential synergism.

Small interfering RNA (siRNA) transfection

siRNAs targeting MAPK12 (si-h/m-Mapk12_001: 5′-CAGCTGAAGGAGATCATGA-3′; si-h/m-Mapk12_002: 5′-GCATCATGGCGGAGATGAT-3′) and a negative control were synthesized by Tsingke Biotechnology. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) according to the manufacturer’s instructions. Cells were seeded into 6-well plates one day prior to transfection. When cell confluence reached 50–60%, siRNAs and transfection reagents were diluted separately in Opti-MEM medium, mixed, and incubated to allow complex formation before being added to the cells. After 48 h of transfection, cells were harvested for subsequent protein expression analysis.

Western blot analysis

Following treatment, total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a bicinchoninic acid (BCA) assay. Equal amounts of protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk or a rapid blocking buffer for 1 h at room temperature, followed by overnight incubation at 4 ℃ with primary antibodies. After washing with Tris-buffered saline with Tween 20 (TBST), membranes were incubated with HRP-conjugated secondary antibodies for 1–2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and imaged with a chemiluminescence detection system. Band intensities were quantified using ImageJ software.

Immunofluorescence staining

Cells were seeded into 24-well plates containing sterile glass coverslips, incubated overnight, and subjected to the indicated treatments. At the specified time points, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 5% goat serum for 30 min. Cells were then incubated with primary antibodies overnight at 4 ℃. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies for 1–2 h at room temperature in the dark. Finally, coverslips were mounted using an anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a confocal laser scanning microscope.

Autophagic flux assay (mCherry-GFP-LC3 reporter system)

Cells were seeded into confocal dishes (6-well) and incubated overnight. The following day, cells were infected with an adenoviral vector encoding the mCherry-GFP-LC3 reporter (Hanbio Biotechnology) and subsequently treated with the indicated compounds. After 48 h, fluorescence signals were observed using a confocal microscope. Yellow puncta (mCherry⁺/GFP⁺) indicate autophagosomes, whereas red puncta (mCherry⁺/GFP⁻) represent autolysosomes. Autophagic flux was evaluated by quantifying the number of red and yellow puncta.

Flow cytometric analysis of apoptosis

Following drug treatment, both adherent and floating cells were collected, washed with PBS, and resuspended in 1× binding buffer. For each 100 μL cell suspension, 5 μL Annexin V-fluorescein isothiocyanate (V-FITC) was added, followed by incubation for 15 min at room temperature in the dark. Prior to analysis, 1 μL propidium iodide (PI) staining solution was added, and samples were gently mixed and immediately analyzed using a flow cytometer. Data were analyzed using FlowJo software, and cells were classified as viable, early apoptotic, late apoptotic, or necrotic based on Annexin V and PI staining profiles.

Establishment of drug-resistant cell lines

OS-resistant HCC827 cells were established using a stepwise dose-escalation method. Parental cells were initially exposed to a low concentration of OS (e.g., 10 nM) and passaged every 2–3 days. When cell confluence reached approximately 80%, the culture medium was replaced with fresh medium containing the same drug concentration. Once cells exhibited stable proliferation under this condition, the drug concentration was gradually doubled, and the adaptation process was repeated until cells were able to grow stably in 2 μM OS. The half-maximal inhibitory concentration (IC₅₀) values of resistant and parental cells were determined using the CCK-8 assay, and the resistance index was subsequently calculated.

Animal experiments

All animal experiments were performed under a project license (No. 20230313010) granted by the Animal Ethics Committee of Sichuan University, in compliance with institutional guidelines for the care and use of animals. All experimental mice were housed in a specific pathogen-free (SPF) grade laboratory. Routine husbandry was conducted by qualified professional laboratory animal staff: cage bedding was replaced every other day, and sterile feed and drinking water were replenished twice a week. Throughout the experimental period, investigators monitored the body weight and general health status of all mice every other day to ensure animal welfare. HCC827 parental or drug-resistant cells in the logarithmic growth phase were resuspended and mixed 1:1 with Matrigel, and a total of 3×10⁶ cells were subcutaneously injected into the right axillary region of 4-week-old female BALB/c nude mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). When tumor volumes reached approximately 150–200 mm³, mice were randomly assigned to four groups (n=10 per group): control, PFD monotherapy (100 mg/kg, administered daily by oral gavage), OS monotherapy (5 mg/kg, administered every other day by oral gavage), and combination therapy. Tumor length and width were measured every other day, and tumor volume was calculated using the formula:

Volume=longestdimension×(shortestdimension)22[1]

Statistical analysis

All data were analyzed and visualized using GraphPad Prism software. Results are presented as the mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using an unpaired Student’s t-test, while comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). A P value <0.05 was considered statistically significant (P<0.05, P<0.01, P<0.001). All experiments were independently repeated at least three times.

Results

Acquired resistance to first-line OS in patients with EGFR-mutant NSCLC

To characterize the current landscape of resistance to EGFR-TKI-targeted therapy in clinical practice, we conducted a retrospective analysis of 201 patients with EGFR-mutant NSCLC who received first-line OS treatment at West China Hospital, Sichuan University, between 2019 and 2024 (Table 1). Among the enrolled patients, 68.67% were female, with a median age of 64 years. The majority of patients (95.02%) were diagnosed with stage IV NSCLC. The predominant EGFR mutation subtypes were exon 19 deletions (56.22%) and the exon 21 L858R point mutation (40.80%).

During the median follow-up period, 59.70% of patients experienced disease progression after OS treatment, and the median PFS was only 16 months. These findings indicate that acquired resistance to OS remains a major barrier to durable clinical benefit in patients with EGFR-mutant NSCLC. Therefore, developing effective sensitization strategies and overcoming acquired resistance to EGFR-TKIs represent critical clinical priorities.

PFD synergizes with OS/AF to exert potent antitumor effects in NSCLC cells

To investigate whether PFD enhances the cytotoxic effects of OS or AF in NSCLC cells, we first performed a series of in vitro functional assays to systematically evaluate the antitumor activity of the combination treatments. As shown in Figure 1A-1D and Figure S1A,S1B, in three EGFR-mutant NSCLC cell lines (PC9, HCC827, and H1975), PFD dose-dependently potentiated the antiproliferative effects of OS/AF. Notably, increasing concentrations of PFD markedly enhanced the cytotoxic efficacy of OS/AF against tumor cells. Consistently, colony formation assays further confirmed these findings (Figure 1E,1F and Figure S1C,S1D). Compared with PFD or OS/AF monotherapy, the combination treatment significantly suppressed the clonogenic capacity of NSCLC cells. While single-agent treatments exerted only modest inhibitory effects on colony formation, the combination regimen led to a pronounced reduction in both colony number and colony size.

Figure 1 PFD synergizes with OS/AF against NSCLC in vitro and in vivo. (A-D) Relative survival rate of PC9 (A,C) and HCC827 (B,D) cells treated with gradient concentrations of PFD combined with OS (A,B) or AF (C,D), detected by CCK-8 assay. (E,F) Colony formation capacity of PC9 (E) and HCC827 (F) cells treated with PFD alone, OS/AF alone, or their combination, with quantitative analysis of relative clone area on the right. (G,H) Bliss synergy scores and dose-effect matrix heatmaps of PFD combined with AF or OS in PC9 (G) and HCC827 (H) cells, analyzed via SynergyFinder platform. (I) Representative images of dissected tumors from each group. (J) Tumor growth curves during the treatment period. (K) Statistical analysis of tumor weight at the experimental endpoint. **P<0.01, ***P<0.001 vs. control or monotherapy group. AF, afatinib; CCK-8, Cell Counting Kit-8; NSCLC, non-small cell lung cancer; OS, osimertinib; PFD, pirfenidone.

To further characterize the interaction between PFD and OS or AF, we employed the SynergyFinder platform to evaluate drug combination effects based on the Bliss independence model. As shown in Figure 1G,1H and Figure S1E,S1F, the Bliss synergy scores for the PFD and OS combination in PC9, HCC827, and H1975 cells were 22.815, 18.447, and 16.347, respectively. Similarly, the Bliss synergy scores for the PFD and AF combination were 21.875, 19.705, and 14.974 in the respective cell lines. Importantly, all synergy scores exceeded 10 across the tested cell lines, indicating that the interactions between PFD and OS/AF are not merely additive but exhibit significant synergistic antitumor effects.

To further validate the in vivo antitumor efficacy of the combination therapy, we established a subcutaneous xenograft model using HCC827 cells in nude mice and evaluated the therapeutic effect of PFD in combination with OS. As shown in Figure 1I, monotherapy with either PFD or OS resulted in only modest inhibition of tumor growth derived from HCC827 cells. In contrast, the combination treatment markedly suppressed tumor progression, as evidenced by a significantly reduced tumor growth rate. At the experimental endpoint, both tumor volume and tumor weight in the combination group were significantly lower than those in the control and monotherapy groups (Figure 1J,1K). These findings demonstrate that PFD significantly enhances the antitumor efficacy of OS in vivo, providing robust preclinical evidence to support the potential clinical translation of this combination strategy.

PFD combined with OS/AF suppresses the PI3K/AKT/mTOR signaling pathway in NSCLC cells

To elucidate the molecular mechanisms underlying the synergistic antitumor effects of PFD in combination with OS or AF, we performed transcriptomic sequencing of PC9 cells treated with control, OS/AF monotherapy, or PFD plus OS/AF (Figure 2A,2B). Compared with the AF monotherapy group, combination treatment resulted in 2,403 upregulated and 2,280 downregulated differentially expressed genes (DEGs). Similarly, relative to the OS monotherapy group, 1,679 genes were upregulated, and 1,357 genes were downregulated following combination treatment. These findings suggest that the combination therapy induces a distinct transcriptional regulatory profile compared with EGFR-TKI monotherapy. We hypothesized that the synergistic cytotoxic effects of the combination treatment may be associated with dysregulation of key downstream survival and proliferation pathways of EGFR. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs revealed that, compared with OS/AF monotherapy, the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway was significantly enriched and downregulated following PFD combination treatment (Figure 2C,2D), indicating that this pathway may represent a critical downstream target mediating the sensitizing effects of PFD on EGFR-TKIs. Based on the KEGG enrichment results, we further extracted DEGs involved in the PI3K/AKT signaling pathway and generated clustered heatmaps to visualize gene expression patterns (Figure 2E,2F). The heatmap analysis demonstrated that, in both AF+PFD (Figure 2E) and OS + PFD (Figure 2F) treatment groups, key functional genes involved in cell proliferation, anti-apoptosis, and cell cycle progression—such as PIK3CA, AKT1, mTOR, BCL2, and CCND1—were markedly downregulated. In contrast, a subset of negative regulators within the pathway exhibited upregulated expression. Collectively, these transcriptomic data indicate that PFD in combination with OS/AF systematically suppresses PI3K/AKT signaling at the genome-wide level, providing a robust molecular basis for subsequent validation at the protein level.

Figure 2 PFD combined with OS/AF downregulates the PI3K/AKT/mTOR signaling pathway in NSCLC cells. (A,B) Volcano plots of DEGs from transcriptome sequencing in PC9 cells treated with AF vs. PFD + AF (A) and OS vs. PFD + OS (B), with the screening threshold set as Padj<0.05 and |log2FoldChange| >0. (C,D) KEGG pathway enrichment bubble plots of DEGs from AF vs. PFD + AF (C) and OS vs. PFD + OS (D) groups, with the PI3K-AKT signaling pathway highlighted. (E,F) Clustering heatmaps of DEGs enriched in the PI3K/AKT signaling pathway from AF vs. PFD + AF (E) and OS vs. PFD + OS (F) groups. (G-J) Western blot analysis of total and phosphorylated protein levels of mTOR, PI3K and AKT in PC9 (G,H) and HCC827 (I,J) cells treated with PFD alone, OS/AF alone, or their combination, with GAPDH as the loading control. (K,L) Relative survival rate of HCC827 (K) and PC9 (L) cells treated with gradient concentrations of mTOR inhibitor Rapamycin combined with OS or AF, detected by CCK-8 assay. AF, afatinib; AKT, protein kinase B; CCK-8, Cell Counting Kit-8; DEG, differentially expressed gene; mTOR, mammalian target of rapamycin; NSCLC, non-small cell lung cancer; OS, osimertinib; PFD, pirfenidone; PI3K, phosphatidylinositol 3-kinase.

To validate the transcriptomic findings, we performed Western blot analysis to assess the total protein expression and phosphorylation levels of key components in the PI3K/AKT/mTOR signaling pathway. As shown in Figure 2G,2H and Figure S2A,S2B, in PC9 cells, treatment with PFD or OS/AF alone exerted minimal or only modest inhibitory effects on the total and phosphorylated levels of PI3K, AKT, and mTOR. In contrast, combination treatment with PFD and OS/AF markedly reduced the expression levels of PI3K, phospho-PI3K (Tyr458), AKT, phospho-AKT (Ser473), mTOR, and phospho-mTOR (Ser2448), indicating effective blockade of PI3K/AKT/mTOR pathway activation. These findings were further validated in HCC827 cells (Figure 2I,2J and Figure S2C,S2D). To further determine the functional significance of PI3K/AKT/mTOR pathway inhibition in sensitizing EGFR-TKIs, we treated NSCLC cells with the mTOR-specific inhibitor rapamycin and evaluated its impact on the cytotoxic effects of OS/AF. As shown in Figure 2K,2L, rapamycin dose-dependently enhanced the antiproliferative effects of OS and AF in PC9 and HCC827 cells. These results demonstrate that inhibition of the PI3K/AKT/mTOR pathway effectively sensitizes NSCLC cells to EGFR-TKIs, highlighting its central role in mediating the synergistic antitumor effects of PFD in combination with OS/AF.

PFD enhances the antitumor efficacy of EGFR-TKIs by promoting intact autophagic flux

Autophagy is a critical process regulating tumor cell survival and death under stress conditions, and the PI3K/AKT/mTOR pathway serves as a central negative regulator of autophagy (24). Based on this, we hypothesized that, in addition to directly inhibiting pro-survival signaling pathways, the combination of PFD with OS or AF may exert synergistic cytotoxic effects through modulation of autophagy. To test this hypothesis, we first performed Western blot analysis to assess the expression of autophagy-related markers in NSCLC cells following monotherapy or combination treatment. Additionally, bafilomycin A1 (Baf A1), an inhibitor of autophagosome-lysosome fusion, was used to distinguish between autophagosome formation and degradation. As shown in Figure 3A-3D and Figure S2E-S2H, in the absence of Baf A1, PFD combined with OS/AF led to a slight decrease in LC3B levels in PC9 and HCC827 cells, while the autophagy substrate p62 showed no significant change. In contrast, upon Baf A1 treatment, which blocks autophagosome-lysosome fusion, the combination group exhibited a marked accumulation of LC3B and p62 compared with monotherapy groups. Meanwhile, the expression levels of autophagy initiation-related proteins, including Beclin-1 and ATG5, remained largely unchanged across all groups. Collectively, these findings suggest that PFD combined with OS/AF does not activate autophagy through the canonical Beclin-1/ATG5-dependent pathway. Instead, it directly promotes autophagosome formation while maintaining intact autophagic degradation, thereby enhancing overall autophagic flux.

Figure 3 PFD combined with OS/AF promotes autophagic flux to enhance the cytotoxicity of EGFR-TKIs in NSCLC cells. (A-D) Western blot analysis of autophagy-related proteins (P62, Beclin-1, ATG5, LC3B) in PC9 (A,C) and HCC827 (B,D) cells treated with PFD alone, OS/AF alone, or their combination, with or without the autolysosome inhibitor bafilomycin A1. GAPDH was used as the loading control. (E,F) Immunofluorescence staining of LC3B (green) with DAPI nuclear counterstain (blue) in PC9 cells treated with AF (E) or OS (F) in the presence or absence of PFD. (G,H) Autophagic flux detection in PC9 cells via mCherry-GFP-LC3 tandem fluorescent reporter system, after treatment with OS (G) or AF (H) combined with PFD, with or without bafilomycin A1. (I) Quantitative analysis of autophagosomes (GFP + mCherry + dots) and autolysosomes (mCherry + only dots) per cell from the images in (G,H). (J,K) Relative survival rate of PC9 (J) and HCC827 (K) cells treated with gradient concentrations of autophagy activator Torin 2 combined with OS or AF, detected by CCK-8 assay. AF, afatinib; CCK-8, Cell Counting Kit-8; DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSCLC, non-small cell lung cancer; OS, osimertinib; PFD, pirfenidone; TKI, tyrosine kinase inhibitors.

To directly visualize the regulatory effects of the combination treatment on autophagic flux, we performed immunofluorescence staining to examine the intracellular distribution and expression of LC3B and employed the mRFP-GFP-LC3 dual-fluorescence reporter system to dynamically monitor autophagic flux. Immunofluorescence analysis demonstrated that (Figure 3E,3F), compared with monotherapy groups, cells treated with PFD combined with OS/AF exhibited a significant increase in both the number and intensity of LC3B puncta, consistent with the Western blot results. Furthermore, the mRFP-GFP-LC3 assay provided additional confirmation (Figure 3G-3I). In the absence of Baf A1, the number of red-only puncta (representing autolysosomes) was markedly increased in the combination group compared with monotherapy groups, whereas the number of yellow puncta (representing autophagosomes) remained largely unchanged. Upon Baf A1 treatment, which blocks autophagic degradation, both red and yellow puncta accumulated significantly in the combination group. Collectively, these results demonstrate that PFD combined with OS/AF simultaneously promotes autophagosome formation and autophagosome-lysosome fusion and degradation, thereby enhancing intact autophagic flux in NSCLC cells.

To determine the functional significance of enhanced autophagic flux in the antitumor effects of the combination therapy, NSCLC cells were treated with the autophagy activator Torin 2, and its impact on the cytotoxic efficacy of OS or AF was evaluated. As shown in Figure 3J,3K, torin 2 dose-dependently enhanced the antiproliferative effects of OS/AF in both PC9 and HCC827 cells. These findings indicate that activation of autophagic flux effectively sensitizes NSCLC cells to EGFR-TKIs and represents an additional mechanism underlying the synergistic antitumor effects of PFD combined with OS/AF. Furthermore, rescue experiments were performed to assess the involvement of other cell death pathways. Treatment with the pyroptosis modulator birinapant (Figure S3A-S3D) or the ferroptosis inhibitor ferrostatin-1 (Figure S3E-S3H) failed to reverse the cytotoxic effects of the combination therapy. Flow cytometric analysis revealed that apoptosis contributed to the cytotoxicity of the combination treatment only in a subset of cell lines (Figure S3I). Collectively, these results suggest that ferroptosis and pyroptosis are not the primary modes of cell death induced by the combination treatment, while apoptosis plays only a partial role. In contrast, enhanced autophagic flux represents a central non-apoptotic mechanism by which PFD sensitizes NSCLC cells to EGFR-TKIs.

p38γ is a key driver of acquired resistance to OS

Acquired resistance to OS remains a major challenge in the clinical management of EGFR-mutant NSCLC. Previous studies have identified p38γ as a potential molecular target of PFD, and its aberrant activation has been closely associated with NSCLC progression and resistance to EGFR-TKIs (25). Based on this evidence, we hypothesized that prolonged exposure to OS may induce molecular reprogramming in tumor cells, leading to the emergence of stable resistant clones, a process potentially driven by aberrant activation of p38γ. To test this hypothesis, we established an OS-resistant HCC827 cell line (HCC827-OR) and validated the resistant phenotype using a CCK-8 assay. As shown in Figure 4A, the IC₅₀ of OS in parental HCC827 cells was 38.54 nM, whereas it increased to 10.12 μM in HCC827-OR cells, corresponding to a resistance index of 262.58. Morphological analysis under bright-field microscopy revealed that, compared with parental cells, resistant cells exhibited pronounced mesenchymal-like features, which may indicate a more stable proliferative phenotype (Figure 4B). These results confirm the successful establishment of a stable OS-resistant cell line, providing a reliable model for subsequent investigations into resistance mechanisms and reversal strategies.

Figure 4 p38γ mediates OS acquired resistance via activating PI3K/AKT/mTOR signaling, while autophagic flux impairment in resistant cells is independent of p38γ. (A) CCK-8 assay for the sensitivity of HCC827 parental cells and HCC827 OR cells to OS, with the IC₅₀ fitted by GraphPad Prism. (B) Bright-field morphological images of HCC827 parental cells and HCC827 OR cells. (C) Western blot analysis of basal protein levels of p38γ, total and phosphorylated mTOR, PI3K and AKT in HCC827 and HCC827 OR cells, with GAPDH as the loading control. (D,E) Western blot analysis of p38γ expression in HCC827 and HCC827 OR cells treated with or without OS (D), and in PC9 and HCC827 cells treated with gradient concentrations of OS or AF (E). (F-H) Western blot analysis of total and phosphorylated mTOR, PI3K, AKT and p38γ in HCC827 OR (F), PC9 (G) and HCC827 (H) cells transfected with negative control siRNA (si-NC) or si-MAPK12. (I-K) Colony formation assay of PC9 (I), HCC827 (J) and HCC827 OR (K) cells transfected with si-NC or si-MAPK12, followed by OS or AF treatment, with quantitative analysis of relative clone area shown on the right. (L) Western blot analysis of p38γ and autophagy-related proteins (P62, Beclin-1, ATG5, LC3B) in HCC827 and HCC827 OR cells, with or without bafilomycin A1 treatment. (M) Western blot analysis of p38γ and autophagy-related proteins (P62, LC3B) in HCC827 OR cells transfected with si-NC or si-MAPK12, treated with or without OS and bafilomycin A1. *P<0.05, ***P<0.001. AF, afatinib; AKT, protein kinase B; CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IC50, half maximal inhibitory concentration; mTOR, mammalian target of rapamycin; ns, not significant; OS, osimertinib; PI3K, phosphatidylinositol 3-kinase.

Building upon our previous observation that p38γ expression is upregulated following OS/AF treatment, we further hypothesized that sustained aberrant activation of p38γ and its downstream signaling pathways may represent a key molecular event driving acquired resistance to OS. To test this hypothesis, we performed Western blot analysis to examine the expression levels of p38γ and components of the PI3K/AKT/mTOR signaling pathway in parental HCC827 cells and HCC827-OR. As shown in Figure 4C and Figure S4A, p38γ was markedly overexpressed in HCC827-OR cells compared with parental cells. In parallel, the phosphorylation levels of key proteins within the PI3K/AKT/mTOR pathway were significantly increased, whereas total protein levels remained largely unchanged. These findings suggest that aberrant upregulation of p38γ, together with hyperactivation of the PI3K/AKT/mTOR signaling pathway, may be closely associated with the therapeutic response to EGFR-TKIs and the development of acquired resistance.

To determine whether EGFR-TKI treatment directly regulates p38γ expression, we further examined the changes in p38γ levels in parental and resistant cells following OS treatment, as well as the dose-dependent effects of OS/AF on p38γ expression in sensitive NSCLC cells. As shown in Figure 4D and Figure S4B, OS treatment significantly induced the upregulation of p38γ in parental HCC827 cells. In contrast, HCC827-OR cells maintained persistently high levels of p38γ expression regardless of OS exposure. Furthermore, dose–response experiments demonstrated that both OS and AF induced p38γ expression in a concentration-dependent manner in PC9 and HCC827 sensitive cells (Figure 4E and Figure S4C,S4D). These findings indicate that EGFR-TKI treatment can directly upregulate p38γ expression, and sustained overexpression of p38γ may represent an early adaptive event that enables tumor cells to evade drug-induced cytotoxicity, ultimately contributing to the development of acquired resistance.

To investigate the regulatory role of p38γ in the PI3K/AKT/mTOR signaling pathway, we performed siRNA-mediated knockdown of mitogen-activated protein kinase 12 (MAPK12) (p38γ) in HCC827-OR resistant cells as well as PC9 and HCC827 sensitive cells and assessed changes in the activation status of key pathway components (Figure S4E). As shown in Figure 4F and Figure S4F, in HCC827-OR cells, silencing of p38γ markedly reduced the phosphorylation levels of PI3K, AKT, and mTOR, which were otherwise aberrantly activated, while total protein levels remained largely unchanged. Consistent results were observed in PC9 (Figure 4G and Figure S4G) and HCC827 (Figure 4H and Figure S4H) sensitive cells. These findings demonstrate that the PI3K/AKT/mTOR pathway functions as a downstream effector of p38γ, and that its sustained hyperactivation in resistant cells is dependent on p38γ overexpression. To further determine whether targeting p38γ modulates sensitivity to EGFR-TKIs, we performed colony formation assays to evaluate the impact of p38γ knockdown on the cytotoxic effects of OS/AF. In PC9 cells, silencing p38γ significantly enhanced the inhibitory effects of OS and AF on colony formation, as evidenced by a marked reduction in relative colony area compared with the negative control group (Figure 4I). This effect was consistently observed in parental HCC827 cells (Figure 4J). Notably, even in highly resistant HCC827-OR cells, p38γ knockdown substantially restored the inhibitory effect of OS on clonogenic growth (Figure 4K). Collectively, these functional data identify p38γ as a critical driver regulating EGFR-TKI sensitivity and mediating acquired resistance to OS at the cellular level.

In addition to p38γ-mediated pathway activation, dysregulation of autophagic flux has also been recognized as a critical mechanism underlying therapeutic resistance in tumor cells (26). Therefore, we further examined the autophagic phenotype in parental HCC827 cells and OS-resistant HCC827-OR cells. To distinguish between autophagosome formation and degradation, Baf A1, an inhibitor of autophagosome–lysosome fusion, was utilized. As shown in Figure 4L and Figure S4I, in the absence of Baf A1, HCC827-OR cells exhibited significantly higher basal levels of LC3B compared with parental cells, accompanied by a modest accumulation of the autophagy substrate p62, while the expression levels of autophagy initiation-related proteins, including Beclin-1 and ATG5, remained unchanged between the two groups. Upon Baf A1 treatment, which blocks autophagic degradation, parental cells showed a pronounced time-dependent accumulation of LC3B and p62, whereas the extent of accumulation in resistant cells was markedly attenuated. These results indicate that OS-resistant cells display a defective autophagic flux phenotype characterized by increased autophagosome formation but impaired autophagosome–lysosome fusion and degradation. This dysregulation may represent an additional mechanism by which resistant cells evade the cytotoxic effects of OS. Finally, to determine whether the autophagic flux blockade in resistant cells is mediated by p38γ, we performed p38γ knockdown in HCC827-OR cells and assessed the expression of autophagy-related markers. As shown in Figure 4M and Figure S4J, silencing p38γ did not significantly alter the levels of LC3B or p62, regardless of OS or Baf A1 treatment, nor did it rescue the impaired autophagic flux in resistant cells. Collectively, these findings demonstrate that autophagic flux regulation in resistant cells is independent of p38γ and represents a distinct resistance-associated molecular event separate from the p38γ/PI3K/AKT/mTOR axis. These results further suggest that PFD may exert its therapeutic effects through dual mechanisms.

Based on our previous findings demonstrating that specific knockdown of p38γ significantly enhances the cytotoxic effects of OS in both sensitive and resistant cells—thereby effectively reversing acquired resistance—primarily through suppression of aberrant activation of the downstream PI3K/AKT/mTOR pro-survival signaling pathway, we next sought to determine whether PFD could recapitulate the biological effects of p38γ downregulation and restore sensitivity of resistant cells to OS. To address this hypothesis, we systematically conducted functional validation and mechanistic investigations of combination treatment in HCC827-OR resistant cells.

To evaluate whether PFD can overcome acquired resistance to OS, we first performed CCK-8 assays to assess the antiproliferative effects of the combination treatment in HCC827-OR cells. As shown in Figure 5A, in the absence of PFD, the IC₅₀ of OS in HCC827-OR cells was 10.68 μM. Notably, as the concentration of PFD increased from 1 mM to 4 mM, the IC₅₀ of OS in resistant cells progressively decreased from 8.475 μM to 3.73 μM, indicating that PFD effectively restores sensitivity to OS. We next quantified the drug interaction using the SynergyFinder platform. As shown in Figure 5B, the combination of PFD and OS yielded a Bliss synergy score of 29.859±2.85 in HCC827-OR cells, which is substantially above the threshold of 10, confirming a strong synergistic antitumor effect in resistant cells. Furthermore, colony formation assays validated the long-term inhibitory effects of the combination treatment (Figure 5C). Compared with the control, PFD monotherapy, and OS monotherapy groups, the combination of PFD and OS nearly completely abolished the clonogenic capacity of HCC827-OR cells, as evidenced by a marked reduction in relative colony area. These results provide direct evidence that the combination treatment potently suppresses the long-term proliferative capacity of resistant cells.

Figure 5 PFD reverses OS acquired resistance in NSCLC cells. (A) Relative survival rate of HCC827 OR cells treated with gradient concentrations of OS alone or in combination with 1, 2, 4 mM PFD, detected by CCK-8 assay, with the IC₅₀ values fitted by GraphPad Prism. (B) Bliss synergy score and dose-effect matrix heatmap of PFD combined with OS in HCC827 OR cells, analyzed via SynergyFinder platform. (C) Colony formation capacity of HCC827 OR cells treated with control (Ctrl), PFD alone, OS alone, or PFD + OS combination, with quantitative analysis of relative clone area shown in (D). (D) Western blot analysis of total and phosphorylated protein levels of mTOR, PI3K and AKT in HCC827 OR cells treated with PFD alone, OS alone, or their combination, with GAPDH as the loading control. (E) Western-blot-based quantification of p-mTOR, p-PI3K, p-AKT and p38γ in HCC827-OR transfected with si-NC or si-MAPK12. Data are presented as mean ± SD. ns, P≥0.05; **, P<0.01. AKT, protein kinase B; CCK-8, Cell Counting Kit-8; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IC50, half maximal inhibitory concentration; mTOR, mammalian target of rapamycin; ns, not significant; NSCLC, non-small cell lung cancer; OS, osimertinib; PFD, pirfenidone; PI3K, phosphatidylinositol 3-kinase; SD, standard deviation.

To elucidate the core molecular mechanism by which PFD reverses acquired resistance, we performed Western blot analysis to examine the activation status of the PI3K/AKT/mTOR signaling pathway following p38γ knockdown in HCC827-OR cells. As shown in Figure 5D-E, compared with the si-NC control group, specific silencing of p38γ significantly reduced the phosphorylation levels of PI3K, AKT, and mTOR in HCC827-OR cells. These results directly demonstrate that targeting p38γ effectively blocks the aberrant and sustained activation of the PI3K/AKT/mTOR pathway in resistant cells. Collectively, these findings identify p38γ as a key upstream regulator of the PI3K/AKT/mTOR signaling cascade and elucidate the central mechanism by which PFD overcomes OS resistance—namely, by targeting p38γ to suppress downstream pro-survival signaling, thereby restoring the sensitivity of resistant cells to OS (Figure 6).

Figure 6 The mechanism of PFD + AF/OS in EGFR resistance cell. PFD reverses OS/AF resistance via two independent pathways: targeting p38γ to block aberrant PI3K/AKT/mTOR pro-survival signaling and enhancing autophagic flux in a p38γ-independent manner. AF, afatinib; AKT, protein kinase B; EGFR, epidermal growth factor receptor; mTOR, mammalian target of rapamycin; OS, osimertinib; PFD, pirfenidone; PI3K, phosphatidylinositol 3-kinase.

Discussion

Synergistic effects of PFD with OS/AF

PFD is an orally administered antifibrotic agent and one of the two therapies approved for the treatment of IPF. Its antifibrotic, antioxidant, and anti-inflammatory activities are thought to arise from inhibition of multiple profibrotic and inflammatory mediators, including TGF-β, matrix metalloproteinases, and proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as from modulation of mitochondrial function and lymphocyte activation (27,28). Given the substantial overlap between fibrotic remodeling and tumor progression, particularly in processes such as extracellular matrix remodeling, chronic inflammation, and fibroblast activation, PFD has attracted increasing attention as a potential anticancer agent. Supporting this possibility, a recent multicentre, randomized phase 2 trial showed that PFD combined with glucocorticoids significantly improved pulmonary function in patients with grade 2–3 radiation-induced lung injury compared with glucocorticoids alone, providing clinical evidence that the therapeutic effects of PFD may extend beyond IPF and may be relevant to cancer-associated pulmonary conditions (29). More importantly, a nationwide population-based study of 9,938 patients with IPF found that PFD use was associated with a 65.3% lower risk of subsequent lung cancer development (adjusted HR, 0.347; 95% CI, 0.258–0.466), with consistent findings in an independent clinical cohort (HR, 0.716; 95% CI, 0.517–0.991) (30). Together, these observations support the possibility that PFD may exert clinically relevant antitumor effects in addition to its established antifibrotic activity.

Accumulating preclinical evidence further suggests that the antitumor effects of PFD involve multiple tumor-associated processes. PFD has been reported to interfere with cancer-associated fibroblast (CAF)-mediated tumor-promoting interactions within the tumor microenvironment, suppress tumor cell proliferation through cell-cycle arrest, promote apoptosis, and inhibit epithelial-mesenchymal transition (EMT), thereby limiting tumor growth and metastatic potential (31). In addition, PFD may modulate several oncogenic signaling pathways, including PI3K/AKT, TGF-β, and MAPK-related pathways, indicating that its anticancer activity is likely mediated through coordinated effects on multiple components of tumor biology rather than through a single molecular target (32-34). Among these mechanisms, the identification of p38γ, a member of the p38 MAPK family, provides a particularly relevant link between PFD and therapeutic resistance. p38γ has increasingly been recognized as a tumor-promoting kinase involved in cancer cell proliferation, survival, stemness maintenance, and resistance to anticancer therapies (33,35). Importantly, p38γ can promote EGFR signaling activity and has been implicated in EGFR-TKI resistance, particularly in KRAS-mutant cancer models. Notably, PFD-mediated inhibition of p38γ has been reported to restore EGFR-TKI sensitivity in resistant models, suggesting that PFD may have potential not only as an anticancer agent but also as a pharmacological sensitizer to targeted therapy (36,37).

These findings provide a rationale for investigating PFD in combination with EGFR-TKIs. Despite the established clinical use of PFD and accumulating evidence of its antitumor activity, whether PFD can enhance the therapeutic efficacy of EGFR-TKIs in EGFR-mutant NSCLC and, importantly, whether it can delay or overcome acquired resistance to OS or AF remain insufficiently defined. Therefore, our study investigated the therapeutic effects of PFD in combination with OS or AF and explored the potential mechanisms underlying their synergistic activity. Our findings provide evidence supporting PFD as a potential adjunctive strategy to improve EGFR-TKI efficacy and overcome therapeutic resistance in EGFR-mutant NSCLC.

Beyond these signaling mechanisms, PFD may enhance EGFR-TKI efficacy through metabolic and tumor microenvironmental regulation. Metabolomic analyses have shown that PFD can reduce lactate and ATP production, thereby suppressing glycolytic activity, while glycolytic reprogramming has been implicated in resistance to third-generation EGFR-TKIs in NSCLC (38,39). Thus, PFD-mediated inhibition of glycolysis may represent an additional mechanism contributing to EGFR-TKI sensitization. Moreover, PFD can suppress fibroblast activation and disrupt CAF-tumor cell crosstalk, which may further attenuate the tumor-promoting microenvironment and contribute to its antitumor effects in vivo (40). Together, these findings suggest that the activity of PFD in combination with EGFR-TKIs may involve coordinated regulation of tumor-intrinsic metabolic programs and the tumor microenvironment.

PFD combined with OS/AF promotes autophagic flux

Autophagy is a dynamic and continuous process in which autophagosomes undergo sequential formation and degradation. Therefore, assessing autophagic activity based on a single stage is insufficient, leading to the introduction of the concept of autophagic flux. Autophagic flux represents a quantitative measure of autophagic activity and reflects the overall turnover rate of the autophagic process over time. This parameter is governed by two key steps: the efficiency of autophagosome formation and the rate of autophagosome–lysosome fusion and subsequent degradation (41). Autophagy is widely recognized to play a dual role in tumor progression, as it can either promote or suppress tumor growth depending on the cellular context and environmental conditions (42).

In this study, we employed Western blotting, immunofluorescence, and a tandem fluorescent autophagy reporter system to demonstrate that PFD enhances autophagic flux by promoting both autophagosome formation and autophagosome–lysosome fusion. This increase in autophagic activity contributes to enhanced cell death in lung cancer cells, including OS-resistant cells, following treatment with OS/AF. Moreover, co-treatment with the autophagy activator torin 2 similarly resulted in increased cell death, further supporting the notion that augmentation of autophagy sensitizes both parental and resistant tumor cells to OS/AF. Collectively, these findings indicate that elevated autophagic flux enhances therapeutic sensitivity and suggest that impaired autophagy may represent a potential mechanism underlying reduced responsiveness to OS. However, the precise molecular targets through which PFD regulates autophagy remain unclear. Whether PFD promotes autophagic flux by modulating mTOR signaling or other autophagy-related pathways requires further investigation, including potential involvement of the janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), nuclear factor-kappa B (NF-κB) (p65), and Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) signaling pathways (43).

p38γ as a potential therapeutic and resistance target

As discussed above, p38γ represents a promising therapeutic target as well as a mediator of drug resistance in lung cancer. In the present study, we found that p38γ expression is upregulated following OS/AF treatment and is markedly overexpressed in OS-resistant cells. Notably, both genetic silencing of p38γ and pharmacological inhibition using PFD significantly enhanced the sensitivity of NSCLC cells, including resistant models, to OS. Furthermore, our data suggest that in resistant cells, p38γ may promote resistance by activating the PI3K/AKT/mTOR signaling pathway, thereby counteracting the therapeutic effects of OS. Taken together, these findings support the notion that p38γ is a promising therapeutic target and a key mediator of acquired resistance in NSCLC.

The association between p38γ overexpression and OS resistance extends beyond the mechanisms described above. Previous studies have reported that p38 MAPK can induce ligand-independent, clathrin-mediated endocytosis, thereby promoting robust internalization of EGFR (36). Notably, clathrin-mediated EGFR endocytosis has been implicated as a key mechanism underlying resistance to gefitinib (44). In addition, exosomes carrying wild-type EGFR can be internalized by EGFR-mutant tumor cells via clathrin-mediated endocytosis. This process leads to reactivation of critical downstream signaling pathways, including PI3K/AKT and MAPK, ultimately contributing to the development of resistance to OS (45). Therefore, activation of p38γ in resistant cells may also promote OS resistance by facilitating EGFR endocytosis, representing an additional potential mechanism linking p38γ to therapeutic failure.

In addition, p38γ has been reported to enhance EGFR transcription through c-Jun–mediated promoter binding and to promote EGFR dephosphorylation via activation of protein tyrosine phosphatase H1 (PTPH1). This dual regulatory mechanism contributes to resistance to EGFR-TKI therapy in KRAS-mutant cells, and pharmacological inhibition of p38γ using PFD has been shown to reverse this phenotype (46). Moreover, p38γ has been demonstrated to activate mTORC1 signaling through phosphorylation of p62 at T269/S272 (47). Consistent with this, our previous studies have also shown that p38γ directly phosphorylates p62 at T269/S272 (48,49), providing a mechanistic basis for the upregulation of mTOR signaling observed in OS-resistant cells.

Role of autophagy in EGFR-TKI resistance

Multiple downstream signaling pathways of EGFR are involved in the regulation of autophagy, including: (I) the EGFR-mTOR pathway, (II) the EGFR-RAS pathway, (III) EGFR-Beclin-1 signaling, (IV) the EGFR-STAT3 pathway, and (V) EGFR-lysosome-associated protein transmembrane 4 beta (LAPTM4B) signaling (50,51). Therefore, it is not surprising that alterations in autophagic activity can be triggered by EGFR-targeted therapies and contribute to the development of drug resistance (52). Compared with other resistance mechanisms, autophagy represents a rapid and broadly conserved cellular response that can be activated within minutes to hours under various stress conditions. Importantly, this process does not require extensive genetic or epigenetic alterations or prolonged selective pressure. Accordingly, autophagy can be regarded as an early and adaptive mechanism by which cancer cells mitigate the cytotoxic stress induced by targeted therapies (53). The role of autophagy in therapeutic resistance appears to be context-dependent and paradoxical. On one hand, enhanced autophagic activity supports tumor cell survival by degrading misfolded proteins and damaged organelles, thereby providing metabolic substrates that facilitate resistance. On the other hand, sustained or excessive activation of autophagy may trigger type II programmed cell death, also known as autophagy-dependent cell death. Despite extensive investigation, it remains unclear under which specific conditions autophagy promotes or suppresses tumor progression and whether it ultimately drives resistance or sensitization to therapy. Further mechanistic studies are therefore required to elucidate how context-specific modulation of autophagic activity governs the development of drug-resistant phenotypes in cancer cells (54,55).

Despite exhibiting higher basal levels of LC3B-II compared with parental cells, OS-resistant cells failed to show further accumulation of LC3B-II following treatment with bafilomycin A1, an inhibitor of autophagosome-lysosome fusion. This finding indicates that autophagic degradation is impaired in resistant cells. Previous studies have suggested that, in addition to lysosomal degradation, autophagosomes can fuse with multivesicular bodies and be secreted into the extracellular space (56). Based on this, it is plausible that in OS-resistant cells, autophagy may shift from a degradative pathway toward a secretory pathway involving fusion with multivesicular bodies. Secretory autophagy represents a noncanonical form of secretion that integrates autophagic machinery with vesicular trafficking processes and relies on conserved autophagy-related components. This process has been increasingly recognized as playing a critical role in tumor progression and therapeutic resistance. While canonical autophagy facilitates intracellular degradation through autophagosome–lysosome fusion, secretory autophagy mediates the extracellular release of specific cargoes and contributes to remodeling of the tumor microenvironment, potentially promoting immune dysregulation and drug resistance (57). Whether secretory autophagy occurs in OS-resistant cells and represents an additional mechanism of resistance warrants further investigation.

Conclusions

In conclusion, this study demonstrates that PFD significantly enhances the efficacy of EGFR-TKIs, and reverses acquired resistance in EGFR-mutant NSCLC. The combination of PFD with OS or AF exhibited strong synergistic antitumor effects in vitro and in vivo, including restoration of sensitivity in OS-resistant models. Mechanistically, PFD improves EGFR-TKI response through dual regulation of p38γ-dependent and p38γ-independent pathways. Specifically, PFD inhibits the p38γ-mediated PI3K/AKT/mTOR signaling pathway, a key driver of EGFR-TKI resistance, while simultaneously activating autophagic flux to enhance tumor cell vulnerability. These findings identify p38γ as a critical mediator of acquired resistance and reveal autophagy activation as an additional mechanism contributing to therapeutic synergy. Given its established clinical use and favorable translational potential, PFD may represent a promising EGFR-TKI sensitizer for overcoming resistance and improving targeted therapy outcomes in NSCLC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0900/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0900/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0900/prf

Funding: This work was supported by the Sichuan Science and Technology Program (No. 2025JDDJ0005), The Fundamental Research Funds for the Central Universities (Nos. SCU2026D017 and 2026qzsyxf06), 1·3·5 Project of State Key Laboratory of Respiratory Health and Multimorbidity, West China Hospital, Sichuan University (Nos. RHM24101 and RHM25101), and The State Key Laboratory of Respiratory Health and Multimorbidity, State Key Laboratory Special Fund (No. 2060204).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0900/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was reviewed and approved by the Biomedical Ethics Review Committee of West China Hospital, Sichuan University [approval No. 2025 Annual Review (2716)] via expedited review. Given the retrospective nature of this study, all clinical data of enrolled patients were anonymized, and no interference with routine clinical diagnosis and treatment was involved during the study. Therefore, the ethics committee granted a waiver of informed consent for this study. All animal experiments were performed under a project license (No. 20230313010) granted by the Animal Ethics Committee of Sichuan University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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