SMARCA4 deficiency contributes to platinum resistance in non-small cell lung cancer by activating the NF-κB-BIRC2/BIRC3 signaling axis

Abstract

Background

SMARCA4-deficient non-small cell lung cancer (NSCLC) is highly aggressive and has a limited response to conventional chemotherapy. The precise mechanisms by which SMARCA4 deficiency contributes to platinum-based chemotherapy resistance in NSCLC remain incompletely understood. This study aims to elucidate this resistance mechanism and provide a theoretical basis for precision treatment in clinical practice.

Methods

The role of SMARCA4 deficiency in NSCLC was analyzed using the cBioPortal database. SMARCA4-knockdown NSCLC cell models were established. Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) and colony formation assays. Apoptosis was tested using flow cytometry. Carboplatin (CBP) sensitivity was evaluated by measuring the half maximal inhibitory concentration (IC₅₀) values. Transcriptomic sequencing was performed to screen underlying platinum resistance mechanisms, which were further validated by quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC). Finally, a xenograft model was established to observe the combined antitumor effects of CBP and potential agents reversing drug resistance.

Results

SMARCA4 deficiency correlated with a poor prognosis for NSCLC patients, and SMARCA4 knockdown promoted malignant phenotypes in NSCLC cell lines. The sensitivity to CBP treatment was attenuated by SMARCA4 downregulation, with concomitant activation of the NF-κB signaling pathway and upregulation of BIRC2 and BIRC3 expression. Bioinformatics analysis confirmed a significant negative correlation between SMARCA4 and BIRC2/BIRC3 messenger RNA (mRNA) expression. Targeted inhibition of NF-κB signaling effectively restored platinum sensitivity in SMARCA4-deficient lung cancer cells and significantly suppressed tumor growth in xenograft models.

Conclusions

SMARCA4 deficiency contributes to platinum resistance in NSCLC by activating the 
NF-κB-BIRC2/BIRC3 signaling axis. Combination therapy with CBP and either an NF-κB inhibitor or an inhibitor of apoptosis proteins (IAP) inhibitor effectively reverses this resistance, providing a novel strategy for the precise treatment of SMARCA4-deficient NSCLC.

Highlight box.

Key findings

• This study identifies SMARCA4 deficiency as a key driver of platinum resistance in non-small cell lung cancer (NSCLC). Mechanistically, it demonstrates for the first time that SMARCA4 deficiency activates the NF-κB-BIRC2/BIRC3 signaling axis, thereby inhibiting tumor cell apoptosis and ultimately inducing carboplatin resistance.

What is known and what is new?

• SMARCA4 deficiency is frequently present in NSCLC and is closely associated with malignant tumor progression, poor prognosis, and chemotherapy resistance. High expression of BIRC2/BIRC3 can mediate tumor apoptosis inhibition and chemotherapy resistance.

• This study demonstrates for the first time that in NSCLC, SMARCA4 deficiency leads to NF-κB activation and high expression of BIRC2/BIRC3.

What is the implication, and what should change now?

• This study elucidates the molecular mechanism of platinum resistance in SMARCA4-deficient NSCLC, providing clear therapeutic targets and a theoretical basis for this high-risk, refractory subtype.

• In clinical practice, patient stratification can be achieved by detecting SMARCA4 deficiency, NF-κB activation, and BIRC2/BIRC3 expression. For platinum-resistant patients with SMARCA4 deficiency, the combination of an NF-κB inhibitor or IAP inhibitor with platinum-based chemotherapy may serve as a novel therapeutic strategy to reverse drug resistance.

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide (1). Non-small cell lung cancer (NSCLC) is the predominant histological subtype, accounting for more than 85% of all cases. NSCLC is characterized by significant molecular heterogeneity, which profoundly influences therapeutic decisions and the prognosis. Although targeted therapies against oncogenic drivers (e.g., EGFR and ALK) and immune checkpoint inhibitors (ICIs) against programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) have transformed treatment paradigms and prolonged the survival of patients with specific molecular subsets of tumors (2-7), they benefit only a minority of patients. Consequently, platinum-based doublet chemotherapy continues to serve as the first-line treatment for patients with advanced NSCLC who lack targetable mutations or who are ineligible for immunotherapy.

The Switch/Sucrose Non-Fermentable (SWI/SNF) complex is a crucial chromatin remodeling complex in eukaryotes that plays a central role in modulating chromatin architecture and gene expression. Composed of 10–15 subunits, the complex utilizes the ATPase activity of its core components (such as SMARCA4 or SMARCA2) to dynamically regulate nucleosome positioning and chromatin accessibility processes that directly influence the transcription of genes involved in cell cycle progression, differentiation, and DNA damage repair (8,9). Dysregulation of SWI/SNF subunits (e.g., through mutation, deletion, or epigenetic silencing) is frequently observed in human cancers. Cancer genome sequencing has revealed that SMARCA4 mutations are relatively common in NSCLC, occurring in approximately 6–10% of cases (10,11). These mutations often result in a loss of function, disrupting SWI/SNF-mediated chromatin remodeling and thereby promoting tumorigenesis, underscoring the critical tumor-suppressive role of SMARCA4 (12,13). However, the mechanisms by which SMARCA4 deficiency drives cancer progression remain incompletely understood and continue to pose a significant research challenge.

SMARCA4 mutations are closely associated with a poor prognosis for lung cancer patients (12,14,15), with a high prevalence of SMARCA4 deficiency observed in NSCLC, particularly in the large cell carcinoma (LCC) subtype (16). However, the majority of patients are diagnosed with SMARCA4-deficient NSCLC at advanced stages, and chemotherapy remains a cornerstone treatment for advanced disease. Accumulating clinical evidence indicates that SMARCA4-deficient NSCLC exhibits inherent resistance to platinum-based agents. Among this patient population, progression-free survival (PFS) and overall survival (OS) are significantly shortened (17,18). Since platinum drugs such as cisplatin and carboplatin (CBP) are standard therapies for lung cancer (19), this resistance poses a major therapeutic challenge for SMARCA4-deficient NSCLC. Studies suggest that SMARCA4 loss of function disrupts epigenetic regulation, impairs DNA damage repair pathways, including homologous recombination, and promotes genomic instability, all of which are intrinsically linked to the chemotherapy response (20,21). Thus, SMARCA4 mutation serves not only as an important marker in tumor biology but also as a key determinant of platinum resistance (22). Additionally, SMARCA4 deficiency often co-occurs with other mutations, such as TP53, KRAS, STK11, and KEAP1 mutations, which have also been implicated in platinum resistance (23-25). Nevertheless, the specific molecular mechanisms underlying SMARCA4 deficiency-mediated resistance to platinum chemotherapy remain unclear, and related clinical translational research is still limited.

Therefore, elucidating the molecular mechanisms by which SMARCA4 deficiency drives platinum resistance and developing novel therapeutic strategies to overcome this resistance are critical for improving the outcomes of patients with this molecular subset of NSCLC. In this study, we aimed to elucidate the molecular mechanisms by which SMARCA4 deficiency affects the response to platinum-based chemotherapy and to further explore novel therapeutic strategies that could restore the sensitivity of SMARCA4-deficient tumors to such chemotherapy, thereby improving the prognosis of patients with SMARCA4-deficient NSCLC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0527/rc).

Methods

Bioinformatics analysis

SMARCA4 mutation data and clinical information were obtained from the Memorial Sloan Kettering-Clinicogenomic, Harmonized Oncologic Real-world Dataset (MSK-CHORD) dataset comprising 7,809 NSCLC patients and The Cancer Genome Atlas (TCGA) PanCancer Atlas dataset including 566 lung adenocarcinoma (LUAD) patients via the cBioPortal database (https://www.cbioportal.org/). Messenger RNA (mRNA) expression data were downloaded from three independent LUAD cohorts: [Clinical Proteomic Tumor Analysis Consortium (CPTAC), Cell 2020, n=110], (TCGA, Nature 2014, n=230), and (TCGA, PanCancer Atlas, n=566). The association between BIRC2/BIRC3 and SMARCA4 mRNA expression was evaluated using Spearman’s correlation analysis.

Publicly available RNA sequencing (RNA-seq) data and clinical annotations of LUAD patients were retrieved from TCGA database. Patients were stratified into high and low expression groups based on the SMARCA4 mRNA levels. Kaplan-Meier survival curves were generated for each group, and OS was compared using the log-rank test. Data processing, survival analyses, and figure generation were performed using R software (version 4.5.1).

Cell culture

The human lung cancer cell lines NCI-H460 (CL-0299) and NCI-H1437 (CL-0631) were provided by Pricella Biotechnology Co., Ltd. (Wuhan, China). The cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 ℃ in a 5% CO₂ incubator.

Antibodies, reagents and compounds

Antibodies against SMARCA4 (1:1,000 dilution, 49360S, Cell Signaling Technology, Boston, USA), β-actin (1:20,000 dilution, HRP-66009, Proteintech, Wuhan, China), NF-κB (1:2,000 dilution, 10745-1-AP, Proteintech, Wuhan, China), pNF-κB (1:2,000 dilution, 80379-2-RR, Proteintech, Wuhan, China), BIRC2 (1:2,000 dilution, 10022-1-AP, Proteintech, Wuhan, China), BIRC3 (1:2,000 dilution, 24304-1-AP, Proteintech, Wuhan, China), and cleaved caspase-3 (1:1,000 dilution, 25128-1-AP, Proteintech, Wuhan, China) were used.

Modified RPMI medium (SH30809.01, Cytiva, Shanghai, China), fetal bovine serum (AC03L055, Life-iLab, Shanghai, China), penicillin-streptomycin solution (AC03L332, Life-iLab, Shanghai, China), trypsin (AC15L821, Life-iLab, Shanghai, China), Annexin V-647/PI apoptosis detection kit (AC12L043, Life-iLab, Shanghai, China), RIPA lysis buffer (P0013B, Beyotime, Shanghai, China), protease inhibitor cocktail (EDTA-free, 100× in DMSO; HY-K0010, MedChemExpress, Shanghai, China), phosphatase inhibitor cocktail I (100× in DMSO; HY-K0021, MedChemExpress, Shanghai, China), puromycin (E607054, Sangon Biotech, Shanghai, China), and a Cell Counting Kit-8 (CCK-8) assay kit (C0005, TargetMol, Shanghai, China) were used.

CBP (S1215, Selleck, Shanghai, China), BAY 11-7082 (S2913, Selleck, Shanghai, China), and AZD5582 (S7362, Selleck, Shanghai, China) were used.

Stable cell lines

Stable SMARCA4-knockdown cell models were established in H460 and H1437 lung cancer cell lines using lentiviral vector-mediated shRNA knockdown technology (Genechem, Shanghai, China). After selection with puromycin, the knockdown efficiency was confirmed by quantitative polymerase chain reaction (qPCR) and Western blot analyses. The sequences of sh-SMARCA4 and the negative control are provided in Table S1.

qPCR

Total RNA was extracted using RNA-easy isolation reagent (R701-01, Vazyme, Nanjing, China). Reverse transcription was performed with HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (R223-01, Vazyme, Nanjing, China) to synthesize cDNA. qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme, Nanjing, China). β-actin was used as the reference gene, and the relative expression of target genes was calculated using the 2^-ΔΔCt method. The primer sequences are listed in Table S2.

Western blot analysis

Cells from each group were collected and lysed using RIPA lysis buffer supplemented with 1% protease inhibitors and 1% phosphatase inhibitors. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (P0010; Beyotime, Shanghai, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in tris-buffered saline with tween-20 (TBST) for 1.5 hours at room temperature, followed by an incubation with primary antibodies overnight at 4 ℃ and subsequently with secondary antibodies for 1.5 hours at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) reagent (MA0186-1, Meilunbio, Dalian, China).

Transcriptome analysis

Total RNA was extracted from H460 cells in both the sh-SMARCA4 group and the negative control (sh-NC) group (n=3) using TRIzol reagent (15596026, Life Technologies, Shanghai, China). Transcriptome sequencing was performed by Shanghai APTBIO Biotechnology Co., Ltd. The raw sequencing data were subjected to quality control using FastQC, followed by alignment to the reference genome using HISAT2. Gene expression levels were quantified with FeatureCounts and reported as FPKM values. For the differential gene expression analysis, read count data were analyzed with DESeq2. Differentially expressed genes (DEGs) were identified using the following criteria: adjusted P value (padj) <0.05 and |log₂(fold change)| >1. A functional enrichment analysis of the DEGs was conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases.

CCK-8 assay

Cells from the sh-SMARCA4 group and the sh-NC group were seeded into 96-well plates at a density of 2000 cells per well. After 24, 48, 72, and 96 hours of culture, 10 µL of CCK-8 reagent was added to each well, followed by an incubation at 37 ℃ for 1 hour. The optical density (OD) at 450 nm was measured using a microplate reader (AMR-100, ALLSHENG, Hangzhou, China).

For drug treatment experiments, H460 (5×10³ cells/well) and H1437 (6×10³ cells/well) cells were plated in 96-well plates and cultured for 24 hours to allow adhesion. After treatment with single or combined drugs for 48 hours, 10 µL of CCK-8 reagent was added to each well and incubated at 37 ℃ for 1 hour. The absorbance at 450 nm was subsequently measured using a microplate reader.

Colony formation assay

H460 and H1437 cells from both the sh-SMARCA4 group and the sh-NC group were seeded into 6-well plates at a density of 300 cells per well, with three replicate wells per group. The cells were cultured under standard conditions for 14 days, after which the medium was changed twice per week. The cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The plates were air-dried and photographed, and the number of colonies was determined using ImageJ software.

Half maximal inhibitory concentration (IC₅₀) assay

H460 and H1437 cells were seeded into 96-well plates at densities of 5×10³ and 6×10³ cells per well, respectively, and cultured for 24 hours to allow adhesion. CBP was serially diluted in culture medium to generate eight concentrations (0, 12.5, 25, 50, 100, 150, 200, 300, and 400 µM). After 48 hours of drug treatment, 10 µL of CCK-8 reagent was added to each well and incubated at 37 ℃ for 1 hour. The absorbance at 450 nm was measured using a microplate reader.

Apoptosis assay

H460 and H1437 cells were seeded in 6-well plates at densities of 2×10⁵ and 3×10⁵ cells per well, respectively, and cultured for 24 hours. After treatment with the indicated drugs for 48 hours, apoptosis was assessed using an Annexin V-647/PI apoptosis detection kit according to the manufacturer’s instructions. The samples were analyzed on a flow cytometer (CytoFLEX LX), and data processing was performed with CytExpert software.

In vivo assay

Female BALB/c nude mice (4 weeks old, SPF-grade) were purchased from Beijing Huafukang Biological Technology Co., Ltd., and housed in a specific pathogen-free (SPF) animal facility under controlled conditions (temperature 22–25 ℃, humidity 50–60%, 12-h light/dark cycle) with free access to food and water. After one week of acclimatization, sh-SMARCA4 H460 cells and sh-NC H460 cells in the logarithmic growth phase were harvested and prepared for injection. Each mouse received a subcutaneous injection of 0.2 mL of the cell suspension (containing 5×10⁶ cells) into the right flank. Animals were allocated to groups by complete randomization using a random number table. Allocation of experimental animals to groups was conducted by an independent investigator and remained concealed. One week after inoculation, tumor-bearing mice in the sh-NC group were randomly divided into two subgroups (n=4) and intraperitoneally injected with either CBP (50 mg/kg, once per week) or normal saline (NS group). The sh-SMARCA4 group was randomly allocated into four subgroups (n=4): NS, CBP, CBP + BAY 11-7082, and CBP + AZD5582. The mice received intraperitoneal injections of the following agents: normal saline (control), CBP (50 mg/kg, once weekly), BAY 11-7082 (3 mg/kg, twice weekly), and AZD5582 (3 mg/kg, twice weekly). The animals were monitored daily during the experiment. Tumor measurements were conducted by an investigator who was blinded to the treatment allocation. Tumor volumes were measured twice per week and calculated using the formula V = (length × width²)/2. After two weeks of treatment, all the mice were euthanized. The tumors were excised, weighed, and photographed. Experiments were performed under a project license (No. 20250619BALB/c-nu20250722226) granted by the Animal Ethics Committee of Qingdao University, in compliance with Qingdao University guidelines for the care and use of animals.

Immunohistochemistry (IHC) assay

The tumor tissues were fixed with 4% paraformaldehyde, subsequently embedded in paraffin and serially sectioned at a thickness of 4 µm. Following deparaffinization and rehydration, antigen retrieval was performed using EDTA buffer (pH 8.0). After cooling, the sections were washed with PBS. Endogenous peroxidase activity was blocked by an incubation with a 3% H₂O₂ solution at room temperature in the dark for 25 minutes. Nonspecific binding sites were blocked with 3% BSA at room temperature for 30 minutes. The sections were then incubated overnight at 4 ℃ with the following primary antibodies: SMARCA4 (GB11258, Servicebio, 1:200, Wuhan, China), Ki67 (GB111499, Servicebio, 1:1,000, Wuhan, China), NF-κB (10745-1-AP, Proteintech, 1:200, Wuhan, China), pNF-κB (80379-2-RR, Proteintech, 1:200, Wuhan, China), BIRC2 (10022-1-AP, Proteintech, 1:200, Wuhan, China), BIRC3 (24304-1-AP, Proteintech, 1:200, Wuhan, China), and cleaved caspase-3 (25128-1-AP, Proteintech, 1:200, Wuhan, China) antibodies. The sections were subsequently incubated with HRP-conjugated secondary antibodies at 37 ℃ for 50 minutes. Color development was achieved using a DAB substrate kit. The nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated through a graded ethanol series, cleared in xylene, and mounted. Sections were scanned using a NanoZoomer slide scanner (NanoZoomer S210, Hamamatsu, Beijing, China) and visualized with NDPView2 software. Three random fields were selected per sample, and the integrated optical density (IOD) was measured using ImageJ software.

Statistical analysis

Data visualization and statistical analysis were performed using GraphPad Prism software (version 9.5.1). Quantitative data are presented as the mean ± standard error of the mean (SEM). Data with normal distribution and equal variance were analyzed by parametric tests. Two-tailed independent sample Student’s t-tests were used to compare the two groups. Comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA). For data not normally distributed or with unequal variance, nonparametric tests (Mann-Whitney U test) were used. A P value <0.05 was considered to indicate statistical significance. All experiments were independently repeated three times. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ns, not significant.

Results

Prevalence and prognostic implications of SMARCA4 mutations in NSCLC

We first analyzed the frequency of SMARCA4 mutations in the MSK-CHORD dataset, which includes 7,809 NSCLC patients, using the cBioPortal database to characterize the mutational profile and clinical significance of SMARCA4 in NSCLC. The results revealed a mutation rate of approximately 8%, with missense and truncating mutations being the most common types (Figure 1A). The survival analysis indicated that patients with SMARCA4 mutations experienced significantly shorter OS than those with wild-type SMARCA4 (17.49 vs. 42.18 months, P<0.05) (Figure 1B). Furthermore, SMARCA4-mutant patients exhibited a lower incidence of targetable molecular alterations. The most frequently co-mutated genes included TP53, STK11, KEAP1, KRAS, and CDKN2A, all of which occurred at significantly higher rates in SMARCA4-mutant patients than in wild-type patients. In contrast, EGFR mutations were more common in the SMARCA4 wild-type group (Figure 1C). Notably, among SMARCA4-mutant patients, the frequency of co-mutations varied considerably: TP53 mutations were the most prevalent (~60%), followed by mutations in KEAP1, STK11, and KRAS, whose mutation rates ranged between 30% and 40%. This distinct mutational landscape involving key driver genes may significantly influence NSCLC tumorigenesis, disease progression, and the treatment response. Previous studies have found that TP53 mutations may modulate platinum response by affecting DNA damage response and apoptosis (26,27). KRAS mutations may promote treatment tolerance through ERK/JNK signaling (25), STK11 mutations may contribute to metabolic deregulation (28), whereas KEAP1 mutations may activate NRF2-mediated antioxidant programs, potentially reducing sensitivity to platinum-induced damage (29).

Figure 1.

SMARCA4 mutation or deficiency is associated with a poor prognosis for patients with NSCLC. (A) Frequency of SMARCA4 mutations in NSCLC patients from the MSK-CHORD dataset (8%, 641/7,809). (B) Comparison of the prognosis between patients with SMARCA4 mutations and patients carrying the wild-type sequence. (C) Co-occurring genetic alterations in patients carrying SMARCA4 mutations and the wild-type sequence. (D) SMARCA4 mutational status and mRNA expression levels in TCGA PanCancer Atlas cohort. (E,F) SMARCA4 mutation is correlated with shorter PFS and OS. (G) Patients with low SMARCA4 mRNA expression have a poor prognosis. *, P<0.05. CI, confidence interval; HR, hazard ratio; NSCLC, non-small cell lung cancer; OS, overall survival; PFS, progression-free survival; TCGA, The Cancer Genome Atlas.

A subsequent analysis of LUAD (TCGA, PanCancer Atlas dataset) revealed that SMARCA4 mutations are associated with reduced mRNA expression, particularly in patients carrying truncating mutations. Notably, a subset of patients without SMARCA4 mutations also exhibited decreased SMARCA4 mRNA levels (Figure 1D). Previous studies have indicated that not all patients with SMARCA4 deficiency harbor SMARCA4 mutations; specifically, only approximately 72.7% (24/33) of SMARCA4-deficient patients carry mutations in this gene (30). Alternative non-mutational mechanisms, such as a loss of heterozygosity (LOH), microRNA-mediated regulation and aberrant splicing of SMARCA4, may also contribute to SMARCA4 deficiency, providing a plausible explanation for the absence of detectable SMARCA4 mutations in a subset of patients with SMARCA4-deficient NSCLC (31).

We further compared survival outcomes between patients with SMARCA4 mutations and those with wild-type SMARCA4 (Figure 1E,1F). The results demonstrated that patients carrying SMARCA4 mutations experienced significantly shorter PFS (17.62 vs. 41.26 months, P<0.05) and OS (34.39 vs. 50.33 months, P<0.05). We evaluated the effect of SMARCA4 mRNA expression levels on patient survival by generating Kaplan-Meier curves using data from TCGA database and compared a high SMARCA4 mRNA expression group (n=428) with a low expression group (n=69) of LUAD patients. The analysis revealed that patients with low SMARCA4 mRNA expression had a significantly worse prognosis (P=0.01) (Figure 1G). These findings suggest that reduced SMARCA4 mRNA expression is a risk factor in LUAD and may serve as a potential prognostic biomarker for LUAD patients.

SMARCA4 deficiency promotes lung cancer cell proliferation and confers resistance to platinum-induced apoptosis

We selected two NSCLC cell lines, H460 and H1437, for this study to investigate the role of SMARCA4 in NSCLC progression. Both cell lines lack SMARCA4 mutations (32) and do not harbor EGFR mutations, consistent with clinical observations indicating mutual exclusivity between SMARCA4 and EGFR mutations. Using lentiviral transduction, we successfully established stable SMARCA4 knockdown models in both cell lines. Subsequent Western blot and qPCR analyses confirmed that both SMARCA4 protein and mRNA expression levels were significantly reduced in the stable knockdown group (sh-SMARCA4 group) compared with the sh-NC group (Figure 2A).

Figure 2.

SMARCA4 knockdown promotes malignant phenotypes in lung cancer cells. (A) SMARCA4 expression levels after lentivirus-mediated knockdown in H460 and H1437 cells. (B) Proliferation curves of H460 and H1437 cells. (C,D) Colony formation assays in H460 and H1437 cells. Staining method: 1% crystal violet; magnification: ×1. (E,F) IC₅₀ values of carboplatin. (G,H) SMARCA4 knockdown attenuated carboplatin-induced apoptosis in H460 (G) and H1437 (H) cells. After treatment with 50 μM CBP for 48 hours, apoptosis was assessed by performing Annexin V/PI staining and flow cytometry. The percentages of apoptotic cells are shown. The data are presented as the mean ± SEM. All experiments were independently repeated three times. *, P<0.05; **, P<0.01; ****, P<0.0001. CBP, carboplatin; IC₅₀, half maximal inhibitory concentration; NC, sh-NC group; OD, optical density; PI, propidium iodide; SEM, standard error of the mean; SH, sh-SMARCA4 group.

We then performed cell proliferation assays to evaluate the effect of SMARCA4 knockdown on the proliferative capacity of NSCLC cells. The results of the CCK-8 assay showed that the proliferation of SMARCA4 knockdown cells was significantly higher than that of the sh-NC cells (Figure 2B). A colony formation assay further confirmed that SMARCA4 knockdown led to significant increases in both the number and size of cancer cell colonies (Figure 2C,2D), indicating that SMARCA4 knockdown markedly increased the long-term proliferative potential of NSCLC cells. We investigated the potential role of SMARCA4 in platinum-based drug resistance in NSCLC cells by conducting additional experiments. After CBP treatment, the IC₅₀ values of SMARCA4 knockdown cells were significantly higher than those of the sh-NC group (H460: 107.6 vs. 38.95 µM, P<0.05; H1437: 178.1 vs. 106.1 µM, P<0.01) (Figure 2E,2F), suggesting a significant reduction in their sensitivity to CBP. Based on the IC₅₀ values, we selected 50 µM as the CBP concentration for treatment. A flow cytometry analysis revealed that after 48 hours of treatment with this concentration, the apoptosis rate was significantly lower in the SMARCA4 knockdown group than in the sh-NC group (H460: 20.73% vs. 38.72%, P<0.01) (Figure 2G). A consistent trend was observed in the H1437 cell line (21.19% vs. 28.13%, P<0.05) (Figure 2H). These results indicate that SMARCA4 knockdown promotes a malignant phenotype in NSCLC cells by increasing their proliferative capacity and reducing their susceptibility to platinum-induced apoptosis.

SMARCA4 deficiency induces platinum resistance via the NF-κB signaling pathway

SMARCA4 deficiency may regulate the expression of multiple genes. We performed RNA-seq on H460 cells with stable SMARCA4 knockdown (sh-SMARCA4 group) and compared them to cells expressing a negative control sequence (sh-NC group) to systematically investigate the effect of SMARCA4 deficiency on the transcriptional profile of lung cancer cells (Figure 3A). The sequencing results revealed 463 DEGs, including 301 upregulated genes and 162 downregulated genes (Figure 3B,3C), indicating that reduced SMARCA4 expression induces substantial changes in the transcriptome of lung cancer cells. The heatmap showed the expression distribution characteristics of the top 120 DEGs across the samples (Figure 3D). The GO enrichment analysis showed that these DEGs were involved in biological processes such as cell adhesion and cell migration (Figure 3E). The KEGG pathway enrichment analysis of the upregulated genes revealed significant enrichment in pathways such as transcriptional misregulation in cancer, the NF-κB signaling pathway, platinum drug resistance, PD-L1 expression and the PD-1 checkpoint pathway in cancer (Figure 3F), all of which are closely associated with tumorigenesis and cancer progression. These findings suggest that SMARCA4 deficiency promotes the activation of the NF-κB signaling pathway.

Figure 3.

SMARCA4 deficiency induced transcriptional changes (RNA-seq). (A) PCA plot. (B) Volcano plot. (C) Differentially expressed genes. (D) Heatmap. (E) GO analysis. (F) KEGG analysis. BP, biological process; CC, cellular component; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; NC, sh-NC group; PCA, principal component analysis; SH, sh-SMARCA4 group.

The KEGG pathway enrichment analysis revealed the significant upregulation of BIRC2 and BIRC3 expression in the platinum drug resistance pathway. Notably, previous studies have established NF-κB as an upstream regulator of both BIRC2 and BIRC3, whereby NF-κB activates their transcription by directly binding to the promoter regions of these genes (33-35). BIRC2 (baculoviral IAP repeat-containing 2, also known as cellular inhibitor of apoptosis protein 1, cIAP1) and BIRC3 (baculoviral IAP repeat-containing 3, also known as cellular inhibitor of apoptosis protein 2, cIAP2) belong to the inhibitor of apoptosis proteins (IAP) family. Both proteins contain baculoviral IAP repeat (BIR) domains and play critical roles in regulating apoptosis, inflammatory responses, and immune signaling pathways (36,37). Functionally, BIRC2 and BIRC3 play overlapping roles and can directly inhibit apoptosis by suppressing the activity of caspase family proteins, such as caspase-3 (38).

Based on these findings, we presume that SMARCA4 deficiency promotes the activation of the NF-κB signaling pathway, leading to increased expression of the BIRC2 and BIRC3 transcripts. The elevated levels of BIRC2/BIRC3 proteins consequently inhibit apoptosis, ultimately contributing to platinum drug resistance in lung cancer cells.

The expression of BIRC2 and BIRC3 is increased in SMARCA4-deficient lung cancers

We examined the expression levels of the key effector molecule p65 (NF-κB) and its phosphorylated form p-p65 (pNF-κB) using Western blot analysis to further validate the activation of the NF-κB signaling pathway following SMARCA4 deficiency. The results demonstrated that the level of p-p65 was significantly increased in SMARCA4-knockdown cells compared with that in the sh-NC group, while the total p65 protein level remained unchanged (Figure 4A). These findings confirm that SMARCA4 deficiency activates NF-κB signaling by promoting the phosphorylation of p65. We investigated the regulatory effect of SMARCA4 deficiency on the expression of the NF-κB downstream molecules BIRC2 and BIRC3 by first measuring their mRNA levels using qPCR. The results showed that both BIRC2 and BIRC3 mRNA expression were significantly upregulated in the SMARCA4-knockdown group compared with the sh-NC group (Figure 4B), consistent with the results of the transcriptomic sequencing data, indicating that SMARCA4 deficiency increases their expression at the transcriptional level. Furthermore, Western blot analysis revealed that the protein levels of BIRC2 and BIRC3 were markedly increased in the SMARCA4-knockdown group (Figure 4A). We assessed the level of cleaved caspase-3, a key marker of apoptotic activity, by performing a Western blot analysis to investigate the role of SMARCA4 deficiency in regulating resistance to platinum drugs in lung cancer cells. The results indicated that the expression level of cleaved caspase-3 was significantly reduced in the SMARCA4-knockdown group compared with the sh-NC group (Figure 4A). These findings suggest that SMARCA4 deficiency decreases the sensitivity of lung cancer cells to platinum-based chemotherapy by inhibiting cleaved caspase-3-mediated apoptosis.

Figure 4.

SMARCA4 deficiency mediates platinum resistance by activating the NF-κB-BIRC2/BIRC3 signaling axis. (A) Western blot analysis of NF-κB (p65), pNF-κB (p-p65), BIRC2, BIRC3, and cleaved caspase-3 levels in SMARCA4-deficient H460 and H1437 cells. (B) qPCR detection of BIRC2 and BIRC3 mRNA expression in SMARCA4-deficient H460 and H1437 cells. (C-E) Correlations between BIRC2/BIRC3 and SMARCA4 mRNA expression in three LUAD cohorts: CPTAC (Cell 2020, n=110; C), TCGA (Nature 2014, n=230; D), and TCGA (PanCancer Atlas, n=566; E). The data are presented as the mean ± SEM. All experiments were independently repeated three times. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. LUAD, lung adenocarcinoma; NC, sh-NC group; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SH, sh-SMARCA4 group; TCGA, The Cancer Genome Atlas.

We further validate the correlation between SMARCA4 and BIRC2/BIRC3 expression in clinical LUAD samples by obtaining three independent datasets from the cBioPortal database and performed a Spearman correlation analysis to evaluate the relationship between their mRNA expression levels. Consistent with the findings from our in vitro experiments, a weak but significant negative correlation was observed between SMARCA4 and BIRC2/BIRC3 in the CPTAC cohort (CPTAC, Cell 2020; n=110) (BIRC2: r=−0.45, P<0.01; BIRC3: r=−0.29, P<0.01) (Figure 4C). A similar negative correlation was detected in TCGA cohort (TCGA, Nature 2014; n=230) (BIRC2: r=−0.24, P<0.01; BIRC3: r=−0.21, P<0.01) (Figure 4D). This inverse relationship was further confirmed in TCGA PanCancer Atlas cohort (n=566) (BIRC2: r=−0.25, P<0.01; BIRC3: r=−0.22, P<0.01) (Figure 4E). These results indicate that elevated expression of BIRC2 and BIRC3 is closely associated with SMARCA4 deficiency, suggesting that BIRC2 and BIRC3 may represent potential therapeutic targets in SMARCA4-deficient lung cancers.

The inhibition of NF-κB signaling or targeting of BIRC2/BIRC3 sensitizes SMARCA4-deficient lung cancer cells to platinum-based chemotherapy

We explored strategies for reversing platinum resistance in SMARCA4-deficient lung cancer by evaluating the antitumor effects of CBP in combination with either the NF-κB inhibitor BAY 11-7082 or the IAP-specific antagonist AZD5582. First, SMARCA4-knockdown H460 cells were treated with CBP combined with BAY 11-7082. After 48 hours, the combination treatment significantly increased the suppression of cell proliferation compared to CBP alone. Further experiments showed that cell viability decreased in a dose-dependent manner after treatment with increasing concentrations of BAY 11-7082. Similarly, in H1437 cells, CCK-8 assays demonstrated that BAY 11-7082 markedly increased the sensitivity of sh-SMARCA4 cells to CBP (Figure 5A,5B). Given that the IAP inhibitor AZD5582 targets the BIR3 domain to inhibit BIRC2/BIRC3 function (39), we further investigated its effect in combination with CBP. CCK-8 proliferation assays revealed that in both SMARCA4-knockdown H460 and H1437 cells, the combination of AZD5582 and CBP resulted in significantly lower cell viability than CBP monotherapy (Figure 5A,5B), indicating a synergistic antiproliferative effect between IAP inhibition and CBP-based chemotherapy.

Figure 5.

Targeted inhibition of NF-κB or IAP effectively restores platinum sensitivity. (A) Combination therapy significantly inhibited proliferation of sh-SMARCA4-transfected H460 cells compared to carboplatin treatment alone (50 μM). (B) Combination therapy significantly inhibited the proliferation of sh-SMARCA4-transfected H1437 cells compared to carboplatin alone (50 μM). (C) Combination therapy (50 μM CBP + 5 μM BAY 11-7082; 50 μM CBP + 5 μM AZD5582) significantly increased apoptosis in sh-SMARCA4-transfected H460 cells compared to carboplatin monotherapy (50 μM). (D) Combination therapy (50 μM CBP + 5 μM BAY 11-7082; 50 μM CBP + 5 μM AZD5582) significantly increased apoptosis in sh-SMARCA4-transfected H1437 cells compared to carboplatin alone (50 μM). (E) Western blot analysis of NF-κB (p65), pNF-κB (p-p65), BIRC2, BIRC3, and cleaved caspase-3 levels in SMARCA4-deficient H460 and H1437 cells following treatment with the NF-κB inhibitor BAY 11-7082. (F) Western blot analysis of BIRC2, BIRC3, and cleaved caspase-3 levels in SMARCA4-deficient H460 and H1437 cells following treatment with the IAP inhibitor AZD5582. The data are presented as the mean ± SEM. All experiments were independently repeated three times. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CBP, carboplatin; IAP, inhibitor of apoptosis proteins.

We subsequently assessed apoptosis rates using flow cytometry. The results showed that in sh-SMARCA4 H460 and H1437 cells, the apoptosis rate was significantly higher in the group treated with CBP combined with BAY 11-7082 than in the group treated with CBP alone (H460: 37.83% vs. 23.96%, P<0.01; H1437: 30.56% vs. 20.39%, P<0.01) (Figure 5C,5D). Similarly, the percentage of apoptotic cells increased from approximately 20% in the CBP monotherapy group to 30% in the group receiving combination treatment with AZD5582 (H460: 34.32% vs. 23.96%, P<0.05; H1437: 29.40% vs. 20.39%, P<0.05) (Figure 5C,5D), further confirming that the drug combinations significantly increased apoptosis in SMARCA4-deficient lung cancer cells.

Mechanistically, Western blot analysis (Figure 5E) revealed that the pharmacological inhibition of NF-κB using BAY 11-7082 in SMARCA4-knockdown cells significantly reduced the protein expression of the key NF-κB transcriptional targets BIRC2 and BIRC3. Concurrently, the level of cleaved caspase-3 was upregulated, indicating the reactivation of the apoptotic pathway. Following treatment with the IAP inhibitor AZD5582 (Figure 5F), BIRC2 and BIRC3 were degraded. This event activated the apoptotic pathway, as evidenced by increased expression of the apoptosis marker cleaved caspase-3. In summary, the IAP inhibitor AZD5582 increases CBP sensitivity in SMARCA4-deficient lung cancer cells by inhibiting BIRC2/BIRC3 and activating the apoptotic pathway. These findings suggest that in SMARCA4-deficient lung cancer cells, the combination of CBP with either an NF-κB inhibitor (BAY 11-7082) or an IAP inhibitor (AZD5582) exerts significantly enhanced antitumor effects.

The inhibition of NF-κB or IAP significantly increases the efficacy of CBP in xenograft mouse models

Given the critical roles of the NF-κB signaling pathway and the IAP BIRC2/BIRC3 in mediating CBP resistance in SMARCA4-deficient lung cancer and considering that the targeted inhibition of these pathways has antitumor efficacy in vitro, we further evaluated whether inhibitors of NF-κB signaling or BIRC2/BIRC3 could increase the efficacy of CBP chemotherapy using a subcutaneous xenograft mouse model of SMARCA4-deficient lung cancer. The NF-κB inhibitor BAY 11-7082 was used to suppress the activity of the NF-κB pathway, whereas AZD5582 was used to inhibit BIRC2/BIRC3 function.

sh-SMARCA4 H460 cells and sh-NC H460 cells were subcutaneously inoculated into nude mice to establish xenograft models. The experiments included six groups with the following design: mice in the sh-NC H460 groups (two groups) were treated with normal saline (vehicle group) or CBP; and mice in the sh-SMARCA4 H460 groups (four groups) were treated with normal saline, CBP alone, CBP combined with BAY 11-7082, or CBP combined with AZD5582. Xenograft tumors derived from sh-SMARCA4 H460 cells (designated the SH group) grew significantly faster than those from sh-NC H460 cells (NC group). In terms of the drug intervention, tumor growth was significantly suppressed in the NC group of mice treated with CBP monotherapy. In contrast, mice in the SH group exhibited marked resistance to CBP treatment, with no significant inhibition of xenograft growth observed. Notably, compared with CBP monotherapy, combined treatment with CBP and the NF-κB inhibitor BAY 11-7082 significantly inhibited the growth of sh-SMARCA4 xenografts (Figure 6A,6B). Similarly, the combination of CBP with the IAP inhibitor AZD5582 led to a significant suppression of tumor growth. Furthermore, the tumor weight measurements were consistent with the observed growth patterns: both combination therapy groups showed significantly lower tumor weights than the CBP monotherapy group (Figure 6C).

Figure 6.

The inhibition of NF-κB or IAP increases the antitumor efficacy of carboplatin in vivo. (A) Tumor growth curves for the six groups. Statistical significance was determined using two-way ANOVA (time, tumor volume). Drug treatment was initiated 7 days after tumor cell inoculation. (B) Representative images of tumors. (C) Tumor weight at the end of the experiment. (D) Immunohistochemical staining was performed to measure the expression of SMARCA4, Ki-67, NF-κB, pNF-κB, BIRC2, BIRC3, and cleaved caspase-3 in xenograft tumor tissues. Magnification: ×400. (E) IOD values of the indicated proteins were quantified using ImageJ software. BAY 11-7082: NF-κB inhibitor. AZD5582: IAP inhibitor. The data are presented as the mean ± SEM; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; CBP, carboplatin; IAP, inhibitor of apoptosis proteins; IOD, integrated optical density; NC, sh-NC group; NS, normal saline; SEM, standard error of the mean; SH, sh-SMARCA4 group.

A subsequent immunohistochemical analysis of tumor tissues revealed that compared with that in the sh-NC group, the Ki-67 expression in tumor tissues in the sh-SMARCA4 group was significantly increased. Furthermore, NF-κB was activated and led to upregulated expression of BIRC2 and BIRC3 and a significant reduction in cleaved caspase-3 levels. In sh-SMARCA4 tumor tissues, the combination therapy (versus CBP monotherapy) resulted in the downregulation of BIRC2/BIRC3 expression, a notable increase in cleaved caspase-3 levels, and a significant decrease in Ki-67 expression (Figure 6D,6E). These findings suggest that the overexpression of BIRC2 and BIRC3 in vivo may play a critical role in the regulation of tumor cell apoptosis. Targeted inhibition of the NF-κB signaling pathway or of BIRC2/BIRC3 may represent a potential therapeutic strategy for reversing platinum-based drug resistance in SMARCA4-deficient lung cancer.

Discussion

SMARCA4, a core ATPase subunit of the SWI/SNF chromatin remodeling complex, is frequently inactivated in NSCLC, where its deficiency is associated with aggressive malignant phenotypes and poor therapeutic responses. Platinum-based agents (such as cisplatin and CBP) remain cornerstone first-line chemotherapies for advanced NSCLC, yet their efficacy is often limited by the emergence of drug resistance. The relationship between SMARCA4 deficiency and platinum resistance, along with its underlying mechanisms, has not been fully elucidated. In this study, we systematically investigated platinum resistance in SMARCA4-deficient NSCLC through a combination of in vitro cellular assays, transcriptomic profiling, validation in clinical cohorts, and in vivo xenograft models. We show for the first time that SMARCA4 deficiency activates the NF-κB-BIRC2/BIRC3 signaling axis, suppresses apoptosis, and thereby confers resistance to CBP. These findings not only indicate the key mechanism by which SMARCA4 regulates chemosensitivity in NSCLC but also provide new directions for precision therapeutic intervention in patients with this subset of NSCLC.

Specifically, transcriptomic sequencing revealed the significant enrichment of the NF-κB signaling pathway and platinum drug resistance pathways in SMARCA4-knockdown lung cancer cells. Western blot analysis further confirmed the increased phosphorylation of the core NF-κB subunit p65 and the concomitant upregulation of its downstream target proteins BIRC2 and BIRC3. Elevated expression of these IAPs suppresses cleaved caspase-3 activity, thereby blocking CBP-induced apoptosis and ultimately leading to chemoresistance. More importantly, multicenter clinical cohorts, including TCGA and CPTAC cohorts, displayed a significant negative correlation between SMARCA4 and BIRC2/BIRC3 mRNA expression in LUAD, thereby bridging our mechanistic findings in vitro with clinical characteristics.

The NF-κB signaling pathway plays a critical role in various physiological and pathological processes, including cell proliferation, apoptosis, and inflammatory responses. Its aberrant activation is closely associated with tumor initiation, progression, and drug resistance (40-42). Members of the IAP family, particularly BIRC2 and BIRC3, function as key negative regulators of apoptotic pathways. Their elevated expression has been linked to platinum-based chemotherapy resistance in multiple cancer types. Specifically, USP35 resists cisplatin-induced apoptosis by stabilizing BIRC3 protein in NSCLC (43). In ovarian cancer, COL11A1 induces the expression of XIAP, BIRC2 and BIRC3 by activating the Src-PI3K/Akt-NF-κB signaling pathway, thereby promoting resistance to cisplatin (44). In this study, we observed that the expression of BIRC2/BIRC3 was upregulated at both transcriptional and protein levels in SMARCA4-deficient cells. After performing specific functional intervention experiments on BIRC2/BIRC3, we found that the expression trends of BIRC2/BIRC3 were consistent with the platinum-resistant phenotype. Therefore, this mechanistically supports that BIRC2/BIRC3-mediated survival signaling may be involved in the platinum resistance process. Notably, SMAC mimetics, which antagonize IAP to restore apoptotic sensitivity, have shown promising potential in preclinical studies of solid tumors (45-47).

This study demonstrated that combination therapy targeting the NF-κB-BIRC2/BIRC3 axis effectively reversed chemoresistance. In vitro, the combination of CBP with either the NF-κB inhibitor BAY 11-7082 or the IAP-specific antagonist AZD5582 significantly reduced the viability of SMARCA4-knockdown cells. Notably, AZD5582 exhibited increased specificity toward BIRC2/BIRC3 because it induced the degradation of BIRC2 and BIRC3. In vivo, the combination of CBP and BAY 11-7082/AZD5582 significantly suppressed tumor growth and reduced the weight of SMARCA4-deficient xenografts in a mouse model.

Several inhibitors targeting IAP have advanced into clinical trials, displaying favorable safety profiles and clinical efficacy (48-50). The findings of this study provide a theoretical basis for the application of these agents to treat SMARCA4-deficient NSCLC. Combination therapies have been widely adopted in oncology because of their ability to target multiple pathways simultaneously, thereby increasing treatment efficacy and reducing the likelihood of drug resistance. Therefore, the use of IAP inhibitors in combination with platinum-based chemotherapy may represent a promising strategy to overcome platinum resistance and improve the prognosis of patients with SMARCA4-deficient NSCLC, and further detection of NF-κB activation or high expression of BIRC2/BIRC3 can effectively identify the population that is more likely to benefit from this combination therapy regimen.

Interestingly, this study revealed that SMARCA4 deficiency upregulated PD-L1-related pathways, suggesting a potential role in modulating antitumor immunity. Further exploration of immune activation mechanisms may increase therapeutic efficacy and provide new strategies to overcome resistance to immunotherapy in SMARCA4-deficient NSCLC. Notably, not all cases of SMARCA4 deficiency were attributable to genetic mutations, suggesting that its expression may also be regulated by epigenetic or posttranscriptional mechanisms, which is consistent with previous reports (30,31).

Nevertheless, this study has several limitations. First, the precise mechanism by which SMARCA4 deficiency regulates the upstream activation of the NF-κB pathway has not been fully elucidated. Future studies should incorporate a chromatin accessibility analysis and other advanced technologies to further elucidate the epigenetic dynamics induced by SMARCA4 deficiency. Second, this study only evaluated CBP among the platinum-based drugs; therefore, the applicability of our findings to other platinum agents remains to be validated. Third, this study was primarily based on NSCLC cell lines and subcutaneous xenograft models, which do not fully recapitulate the complexity of human tumors. Therefore, the mechanism needs to be further validated in patient-derived organoids and patient-derived xenograft models. In addition, the association between NF-κB activation, BIRC2/BIRC3 expression and platinum resistance needs to be verified in large and independent clinical cohorts. Clinically applicable detection methods should be established for patient stratification, and the efficacy of combination therapy regimens should be explored in patients with SMARCA4-deficient NSCLC. Fourth, investigating other potential cooperative mechanisms of drug resistance will be an important focus of future research.

Conclusions

In summary, we revealed that SMARCA4 deficiency promotes platinum resistance through the NF-κB-BIRC2/BIRC3 axis and confirmed that the targeted inhibition of this pathway effectively reverses the drug resistance phenotype. These findings not only provide deeper insights into the pathogenesis of SMARCA4-deficient NSCLC but also suggest a promising combination therapeutic strategy for this patient population.

Supplementary

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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. Experiments were performed under a project license (No. 20250619BALB/c-nu20250722226) granted by the Animal Ethics Committee of Qingdao University, in compliance with Qingdao University guidelines for the care and use of animals.

Data Sharing Statement

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DOI: 10.21037/jtd-2026-0527