TFAP2A induces cisplatin resistance via BNIP3-mediated mitophagy in non-small cell lung cancer

Abstract

Background

Cisplatin resistance in non-small cell lung cancer (NSCLC) significantly limits treatment efficacy. Mitophagy has been shown to play a protective role in cisplatin resistance. This study aims to further investigate the mechanisms underlying this process, with a particular focus on identifying transcription factor AP-2 (activating enhancer binding protein 2) alpha (TFAP2A) as a novel regulatory factor in NSCLC.

Methods

The correlation between TFAP2A and NSCLC was analysed by The Cancer Genome Atlas (TCGA) database and Kaplan-Meier Plotter database, and validated in clinical samples and cisplatin-resistant NSCLC cells (A549-Re and PC9-Re cells). The changes of mitophagy, apoptosis, and proliferation in cisplatin-resistant NSCLC cells were examined by silencing TFAP2A. The regulatory relationship between TFAP2A and BCL2/adenovirus ElB 19kDa interacting protein 3 (BNIP3) was determined by constructing BNIP3 mutations in vitro. Cisplatin-resistant NSCLC cells with different treatments were ectopically grown with rapamycin (RAPA) interventions, and the tumor growth rate was examined in vivo.

Results

Our results suggested that TFAP2A might induce cisplatin resistance via BNIP3-mediated mitophagy in NSCLC. The cell counting kit-8 (CCK-8) assay and colony formation showed that silencing TFAP2A dramatically inhibited proliferation and migration of cisplatin-resistant NSCLC cells. The propidium iodide (PI) staining and MitoSOX staining experiments revealed that silencing TFAP2A significantly improved the apoptosis and mitochondrial reactive oxygen species (ROS) levels in cisplatin-resistant NSCLC cells. Subsequently, western blot, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that TFAP2A silencing induced cell apoptosis and heightened mitochondrial ROS levels through inhibition of mitophagy via inhibiting BNIP3 pathways. Additionally, western blot, colony formation and immunofluorescence experiments demonstrated that TFAP2A silencing reversed RAPA interventions in cisplatin-resistant NSCLC cells. Finally, we confirmed that TFAP2A silencing can inhibit tumor growth in vivo through experiments on xenograft tumor model of nude mice.

Conclusions

TFAP2A overexpresses significantly in NSCLC, and promotes cisplatin resistance through BNIP3-mediated mitophagy

Highlight box.

Key findings

• Transcription factor AP-2 (activating enhancer binding protein 2) alpha (TFAP2A) overexpresses significantly in non-small cell lung cancer (NSCLC), and promotes cisplatin resistance through BCL2/adenovirus ElB 19kDa interacting protein 3 (BNIP3)-mediated mitophagy.

What is known and what is new?

• The clinical application of cisplatin is hampered by the evolving phenomenon of drug resistance. TFAP2A exhibits high expression in various drug-resistant tumors.

• Our findings indicated that high TFAP2A expression is linked to cisplatin resistance in NSCLC cells, and its silencing induces apoptosis, suppresses proliferation, and inhibits mitophagy. BNIP3 is a critical downstream target of TFAP2A, as its mutation disrupts TFAP2A-mediated mitophagy.

What is the implication, and what should change now?

• TFAP2A is a key player in promoting cell proliferation and drug resistance in NSCLC by regulating BNIP3-mediated mitophagy.

• TFAP2A is a therapeutic target to overcome chemotherapy resistance in NSCLC.

Introduction

Lung cancer is recognized as the second most common form of cancer worldwide, accounting for 12.5% of all cancers, posing a significant threat to public health, and is considered the most common cause of death among cancer diagnoses worldwide, accounting for 21% of all cancer deaths (1). Of these, non-small cell lung cancer (NSCLC) contributes to approximately 85% of all lung cancer cases (2). The traditional treatment approaches for NSCLC primarily encompass surgical resection, radiotherapy, and chemotherapy (3). Emerging clinical strategies combining programmed cell death protein 1 (PD-1) inhibitors (pembrolizumab) with chemotherapy have been proven to prolong the overall survival (OS) of patients with advanced NSCLC compared to chemotherapy alone (4). The majority of patients, however, still fail to achieve a durable response to immune checkpoint blockade (ICB) due to intrinsic or adaptive resistance (5). Despite the pivotal role of chemotherapy in NSCLC management, its efficacy has reached a plateau, accompanied by numerous toxic side effects that might compromise patients’ quality of life (6). Over time, cancer cells can develop resistance to chemotherapy drugs, leading to diminished therapeutic outcomes. Consequently, the prognosis for NSCLC remains poor, with a relatively low five-year survival rate (7). Disrupting drug resistance in NSCLC remains a focal point of current clinical research.

Cisplatin is currently a first-line therapeutic agent for NSCLC, which mainly exerts its anticancer effects primarily by inhibiting DNA replication and transcription of tumor cells, resulting DNA strand breaks and miscoding, thereby suppressing the mitotic process (8). However, the clinical application of cisplatin is hampered by the evolving phenomenon of drug resistance.

Transcription factor AP-2 (activating enhancer binding protein 2) alpha (TFAP2A) regulates various cellular processes, including cell growth and tissue differentiation (9). It was reported to be associated with drug resistance in patients with lung adenocarcinoma (LUAD) (10), a predominant type of NSCLC with strong invasive ability and poor prognosis. Elevated levels of TFAP2A were also observed in breast cancer tissues. Notably, patients of triple-negative breast cancer (TNBC) with elevated TFAP2A levels showed shorter overall survival. However, selective silencing of TFAP2A could significantly inhibit the proliferation and migration of TNBC cells (11). More importantly, TFAP2A demonstrates potential roles in prognosis and immunotherapy prediction across various cancers (12). It has also been found to be exhibited with high expression in various drug-resistant tumors (13-15).

Mitophagy represents a crucial cellular process that entails the selective degradation and recycling of damaged mitochondria (16). Cancer cells exhibit a set of common characteristics that include a significant increase in metabolic demand, accelerated cell proliferation, evasion of growth inhibitory signals, resistance to apoptosis, the ability to achieve unlimited replication, induction of neovascularisation, activation of invasive and metastatic mechanisms, and enhanced cellular tolerance to stress (17). These properties require cancer cells to have a large energy supply, as well as the ability to enhance stress tolerance, limit damage and prevent cell death.

Notably, BCL2/adenovirus ElB 19kDa interacting protein 3 (BNIP3) is frequently overexpressed in a variety of tumor samples. However, when assessed on the basis of 5-year survival, no significant association was found between BNIP3 expression levels and the probability of survival in patients with different stages of lung cancer and its subtypes, such as LUAD and lung squamous cell carcinoma (LUSC). However, compelling evidence from two studies demonstrates that high BNIP3 expression predicts poor prognosis in early-stage NSCLC patients (18,19). This suggests that BNIP3-dependent mitophagy and cell survival mechanisms may predominate in the early lung cancer stage, thereby contributing to the development of a more aggressive lung tumor phenotype. Nevertheless, during lung cancer progression, the role of mitophagy becomes more complex with the formation of isolated cell populations. It is both a key factor in enhancing cell survival and may also be a factor in inducing cell death, thus creating some balance in the prognostic role of BNIP3. In addition, in some samples from NSCLC patients, BNIP3 was found to be localized only in the nucleus, which may imply that it is no longer involved in regulating the mitochondria-dependent cell death process, and thus promoting cancer progression (20).

It has been reported that aberrant expression of microtubule-associated protein 1 light chain 3 (LC3), p62, and BNIP3 could impact the autophagic activity of tumor cells, thus affecting tumor growth and metastasis (21). In nude mouse models, BNIP3 was also found to be associated with prognosis of LUSC (22). However, the precise mechanisms by which TFAP2A regulates mitophagy, particularly its potential role in NSCLC, remain largely unknown. In this study, we explored whether TFAP2A promotes NSCLC proliferation by inducing BNIP3-mediated mitophagy. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-646/rc).

Methods

Bioinformatics analysis

A total of 1,041 NSCLC samples and 108 normal samples were downloaded from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) (10). The data format was converted to facilitate the creation of a gene expression matrix. Rigorous data cleaning was conducted to eliminate low-quality genes and samples. Based on clinical data, samples were meticulously screened into paired and unpaired categories. Differential expression analysis was subsequently performed using the R package, limma. Finally, the differential expression analysis results were visually represented using the ggplot2 package in the R package. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

In this study, we utilized the Kaplan-Meier Plotter database (https://kmplot.com/analysis/index.php?p=service&cancer=lung) to investigate the prognostic role of TFAP2A in 2,167 patients with NSCLC. Specifically, we selected mRNA expression data for NSCLC from the database and entered TFAP2A as the target gene for analysis. By clicking the appropriate button, we generated a Kaplan-Meier survival plot depicting the relationship between TFAP2A expression levels and survival rates in NSCLC patients.

Cell culture and intervention

The human NSCLC cell lines utilized in this investigation, specifically A549 and PC9, were acquired from the cell repository of the Chinese Academy of Sciences (https://www.cellbank.org.cn/). Both A549 and PC9 cell lines underwent authentication through short tandem repeat (STR) profiling. Cultivation of all cell lines was performed in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), which was enriched with 10% fetal bovine serum (FBS, Gibco, USA), and maintained at a temperature of 37 ℃ within a 5% CO₂ incubator r (Thermo Scientific, Waltham, MA, USA). To mitigate the risk of bacterial contamination, the culture media were supplemented with 100 IU/mL of penicillin and 100 μg/ml of streptomycin (Beyotime, Shanghai, China). Furthermore, a mycoplasma elimination reagent (Biosharp, Hefei, China) was employed during the cell culture process to avert mycoplasma infections. Routine assessments were carried out using the MycoBlue Kit (FuHeng, Shanghai, China) to confirm the absence of mycoplasma contamination. The cisplatin-resistant NSCLC cells were derived from their sensitive counterparts through incremental induction with escalating concentrations of cisplatin (MCE, Monmouth Junction, NJ, USA).

All cells underwent additional interventions 48 h after modification (via siRNA, shRNA, or plasmid transfection). In this study, 10 nM rapamycin (RAPA) (MCE) was administered for 48 h as part of these interventions.

Gene silencing assay

SiRNAs were purchased from OriGene to target TFAP2A (SR304787), a corresponding scrambled siRNA was used as a control (SR30002). The transfection was conducted at a final siRNA concentration of 50 nM at the predetermined time. Upon achieving 90% confluence, cells were transfected with siRNA segments against TFAP2A (si-TFAP2A-1, si-FAP2A-2 and si-TFAP2A-3) or a non-targeting negative control (si-Control) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s guidelines.

Besides, three pairs of small hairpin RNAs (shRNAs) were used for screening the most effective downregulation of the gene fragment. Then, recombinant lentiviruses with TFAP2A shRNAs and corresponding controls were acquired from Genomeditech (Shanghai, China). These lentiviruses possess a titer of 1×10⁸ transducing units per milliliter (TU/mL). Moreover, some siRNAs were also used in this study as follows: si-TFAP2A-1 was 5'-CGTCTCCGCCATCCCTATTAA-3', si-TFAP2A-2 was 5'-CCCAGATCAAACTGTAATTAA-3', and si-TFAP2A-3 was 5'-TCAGATTGTAGCCATACTTAA-3'.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was extracted from cancer cells using TRIzol reagent t (Ambion, Waltham, MA, USA) by the manufacturer’s instructions. After determining RNA concentration, 500 ng of RNA was reverse-transcribed into cDNA in a 20 µL reaction volume containing M-MLV, adhering strictly to the manufacturer’s guidelines. 1 µL of the resulting cDNA was then mixed with SYBR Green Real-time PCR Master Mix (Vazyme, Nanjing, China), and sense and antisense primers (each at a concentration of 200 nM) to conduct RT-qPCR on Biosystems ViiA 7 (Life Technologies, Waltham, MA, USA). The analysis of gene expression was carried out utilizing the 2^–ΔΔCT method, with the primer sequences sourced from Sangon (Shanghai, China). Primers sequences used were as follows: TFAP2A forward primer was 5'-AGGTCAAT CTCCCTACACGAG-3', and reverse primer was 5'-GGAGTAAGGATCTTGCGACTGG-3'; BNIP3 forward primer was 5'-GCTCCCAGACACCACAAGAT-3', and reverse primer was 5'-TGAGAGTAGCTGTGCGCTTC-3'.

Western blot assay

NSCLC cells were thoroughly lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (V900854-100ML, Vetec, St. Louis, MO, USA) containing protease and phosphatase inhibitors. The supernatants were collected, and the total protein concentrations were determined using a bicinchoninic acid assay (BCA) protein assay (Beyotime). The total proteins were then separated based on their molecular weights on a 10% SDS-polyacrylamide gel. Subsequently, the proteins were transferred horizontally onto polyvinylidene fluoride (PVDF) membranes. After blocking with a 5% bovine serum albumin (BSA) solution, the PVDF membranes were incubated overnight at a low temperature with primary antibodies, including anti-TFAP2A (ab108311; Abcam, Cambridge, UK), anti-β-actin (ab8226; Abcam), anti-microtubule-associated proteins 1A/1B-light chain 3 (LC3I/II) (14600-1-AP; Proteintech, Chicago, IL, USA), anti-sequestosome 1 (p62) (ab109012; Abcam), anti-cytochrome c oxidase subunit IV (COX IV) (ab202554; Abcam), anti-translocase of outer mitochondrial membrane 20 (TOMM20) (ab186735. Abcam, UK), anti-BNIP3 (ab109362; Abcam, UK). Followed by washing membranes three times and incubating with secondary antibodies (ab6721. Abcam UK) for 2 h at room temperature. Under enhanced chemiluminescence (ECL) reagents (Thermo Scientific), the proteins on the PVDF membranes were visualized on a fluorescence imaging system (Amersham Imager 6000, UK). β-actin was used as an internal control.

Cell proliferation assay

Cancer cells were inoculated into 96-well plates at a concentration of 5×10³ cells per well and incubated for a duration of 24 h, followed by treatment with cisplatin at gradient concentrations (1, 10, 100, 500 µg/mL, respectively) for an additional 24 h. A blank control was established by adding 10% cell counting kit-8 (CCK-8) reagent (Bioagrio, Wuhan, China) to a cell-free medium. Optical density (OD) was measured at a wavelength of 450 nm using a microplate reader (SpectraMax Plus384, MD, USA). Cell viability (%) was calculated as (ODtreatment − ODblank)/(ODcontrol − ODblank) × 100%.

Colony formation assay

Cisplatin-resistant NSCLC cells underwent colony formation assays according to international standard guidelines with slight modifications (23). Briefly, cells were plated in 6-well plates at a density of 1,000 cells per well, with 10 µg/mL cisplatin medium exchanged every 4 days, and the experiment was terminated on day 14. After fixation with 4% paraformaldehyde for 30 min, cells were stained with 1% crystal violet (abs817172; Absin, Shanghai, China) for another 10 min, with the dye solution subsequently recovered. Colonies comprising over 50 cells were evaluated using a Leica light microscope (DMil, Leica, GER). The assay was performed in triplicate.

Mito-SOX red staining and flow cytometry analysis

The mitochondrial production of O2⁻ was determined using the mitochondrial superoxide indicator Mito-SOX Red (M36008; Invitrogen). The harvested cells underwent a minimum of three washes with sterile phosphate-buffered saline (PBS) before being treated with a MitoSOX Red solution. In particular, the cells were incubated for 60 minutes at 37 ℃ in a 10 µM MitoSOX Red solution that was formulated in PBS. The fluorescence of MitoSOX Red was subsequently observed using a laser confocal microscope (LSM710, ZEISS, GER).

In the process of flow cytometry analysis, cells underwent a thorough rinsing procedure utilizing PBS on three occasions. Subsequently, they were harvested employing 500 µL of a trypsin-ethylenediaminetetraacetic acid (EDTA) solution. The cells were then subjected to centrifugation at a force of 400 g for a duration of 5 minutes, followed by resuspension in 3 ml of fresh PBS. To ensure the purity of the cell suspensions, they were filtered through a 70 µm Pre-Separation filter. The fluorescence-based flow cytometry analysis was conducted utilizing a BD FACSCantoII flow cytometer, which was equipped with a laser operating at a wavelength of 488 nm. All quantitation was performed using BD FACSDiva software 8. In each experiment, over 20,000 cells per well were analysed. All the results of flow cytometry were further analysed by FlowJo V10.10.0 software.

Luciferase reporter assay

To obtain the promoter sequence of the BNIP3 gene from the UCSC Genome Browser database (http://genome.ucsc.edu/). The upstream 2 kb promoter segment of BNIP3, which harbors a potential binding site for TFAP2A, was inserted into the GV238 luciferase reporter vector (GeneChem, Shanghai, China), resulting in the construct designated as BNIP3-WT. Subsequently, a variant of this segment, in which the TFAP2A binding site was mutated, was also cloned into the same luciferase reporter vector, referred to as BNIP3-Mut. Following this, cells were co-transfected with either BNIP3-WT, BNIP3-Mut, TFAP2A, or an empty plasmid vector for a duration of 48 hours. The luciferase activity was measured using the dual luciferase reporter assay system (Promega, Madison, WI, USA), with firefly luciferase activity being normalized against Renilla luciferase activity.

Immunofluorescence assay

The adherent cisplatin-resistant NSCLC cells on glass slides were fixed with 4% paraformaldehyde (E672002; Sangon, China) after being subjected to various treatments. The propidium iodide (PI) staining solution (ST511; Beyotime, China) fully encompassed the NSCLC cells, and was incubated for 1 h. After discarding the excess staining solution, the 4’,6-diamidino-2-phenylindole (DAPI) staining solution (KGA1808-50; KeyGEN BioTech, China) was added for 5 min. The two colors were independently recorded, and photographed using a fluorescence microscope (DMil, Leica, GER), with at least 5 fields of view captured for each sample.

Cisplatin-resistant NSCLC cells were cultured in 24-well plates. Following a 48 h exposure to various treatments, the cells were incubated with a pre-warmed solution of 0.2 nM mito-tracker green (M7515, Invitrogen, USA) at 37 ℃ for 30 minutes. Afterward, the cells were washed three times with the buffer supplied in the kit, and fixed using 4% paraformaldehyde for a duration of 10 minutes and were then permeabilized with 0.2% Triton X-100. Subsequently, the NSCLC cells were treated with horse serum to prevent nonspecific binding and subsequently incubated overnight at 4 ℃ with the LC3B antibody (ab192890; Abcam, UK). This was followed by a 1 h incubation at room temperature with an Alexa Fluor 594-conjugated secondary antibody (ab150080; Abcam, USA). After the cells were counterstained with DAPI, fluorescence imaging was performed using a laser confocal microscope (LSM710, ZEISS, GER) with at least 5 fields of view captured for each sample.

Xenograft tumor model of nude mice

Healthy male BALB/c nude mice (aged 6-8 weeks, weighing 18–20 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions at a temperature of 25 ℃ and a humidity of 50%, with ad libitum access to food and water. All animal experiments were performed under a project license (No. KS24001) granted by the Ethics Committee of Shanghai Chest Hospital, in compliance with China national or institutional guidelines for the care and use of animals. Before surgery, mice were anesthetized via intraperitoneal injection of 30 mg/kg sodium pentobarbital. Based on the type of A549 cells and the intraperitoneal injected agent, the experiment was divided into four groups (6 per group): sh-Control, sh-TFAP2A, RAPA, and sh-TFAP2A+RAPA. Each mouse received a subcutaneous injection of 2×10⁶ A549 cells (suspended in 200 µL of PBS) into the right flank. One week later, the nude mice were intraperitoneally injected with 5 mg/kg RAPA or an equal volume of solvent once daily for three consecutive weeks. The measurement of tumor volume (volume = length × width²/2) was conducted utilizing calipers at intervals of every 2 to 3 days. After 28 days, all experimental mice were euthanized, and tumor tissue samples were obtained for subsequent pathological analysis. A protocol was prepared before the study without registration.

Histological analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

At the experimental endpoint, tumors from a subset of nude mice were placed in 4% paraformaldehyde, dehydrated through a series of ethanol and xylene solutions, and embedded in paraffin blocks. Subsequently, 5 µm thick sections were prepared. As required by the reagent manufacturer, the sections were stained with hematoxylin and eosin (HE) staining solution (BA-4058. BASO, USA) and then documented under a microscope (DM18, Leica, GER), with a minimum of five fields of view captured per group.

At the experimental endpoint, the transplanted tumors from the nude mice were meticulously excised and processed into 10-micrometer-thick frozen sections. These sections were rinsed three times in PBS and immersed in 0.3% H₂O₂ for 30 min to eliminate endogenous enzymatic and redox activities. The sections were then blocked with horse serum at room temperature for 1 h. Following this, they were incubated with TUNEL staining solution (HY-K1078. MCE, USA) at a 4 ℃ for 1 h. After removing the excess antibody, the sections were ready for observation, with at least five fields of view being captured per group.

Paraffin-embedded specimens were sliced into consecutive sections measuring 4 mm. Following the processes of deparaffinization, rehydration, and washing, these sections were subjected to microwave heating in sodium citrate buffer (P0083, Beyotime, China) for a duration of 30 minutes to facilitate antigen retrieval. Subsequently, the sections underwent a blocking step using an endogenous peroxidase blocking buffer (P0100B, Beyotime, China) for 10 minutes. Following this, a 5% solution of BSA was applied to the sections for 30 minutes to further prevent non-specific binding. The specimens were then incubated overnight at 4 ℃ with primary antibodies targeting Ki-67 (ab15580; Abcam, UK), TFAP2A (ab108311; Abcam, UK), and BNIP3 (ab109362; Abcam, UK). After washing with phosphate-buffered saline (PBS), the sections were stained using an anti-rabbit ICC secondary antibody (A12002-20, NOVO-Light, China) and visualized with a 3,3’-Diaminobenzidine (DAB) substrate kit (K5007, DAKO, DK). Finally, the sections were examined under magnifications of 100× and 200×, with a minimum of five fields of view captured per group, and were counterstained with hematoxylin prior to visualization utilizing a Leica light microscope.

Statistical analysis

Statistical analyses were performed using SPSS 20.0 software, and R packages (ggplot2, v3.4.0) were utilized for data visualization. The results were presented as mean ± standard deviation (SD). Differences between the two groups were analyzed using Student’s t-test. Statistically significant differences among multiple groups were evaluated through one-way analysis of variance (ANOVA), followed by Bonferroni post-hoc tests. A P value less than 0.05 was considered statistically significant.

Results

Increased expression of TFAP2A in cisplatin-resistant NSCLC

Using the TCGA database, we analysed TFAP2A expression in both unpaired (n=1,041) and paired (n=108) NSCLC samples. The results showed a significant up-regulation of TFAP2A in NSCLC tumor tissues compared to normal tissues (Figure 1A). In paired NSCLC samples, a similar pattern was observed, where TFAP2A expression was consistently higher in tumor tissues relative to adjacent normal tissues (Figure 1B).

Figure 1.

Increased expression of TFAP2A in cisplatin-resistant NSCLC. (A) Analysis of TFAP2A expression levels in NSCLC samples using the TCGA database; (B) analysis of TFAP2A expression levels in paired NSCLC patient samples using the TCGA database; (C) Kaplan-Meier plotter database analysis of the difference of the survival between high and low expression of TFAP2A in NSCLC; (D,E) expression levels of TFAP2A mRNA (D) and protein (E) in cisplatin-sensitive and cisplatin-resistant NSCLC cell lines were detected by RT-qPCR and western blot assays, respectively. Data were reported as mean and SD. A P value less than 0.05 was considered to indicate a statistically significant difference. CI, confidence interval; HR, hazard ratio; mRNA, messenger RNA; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TCGA, The Cancer Genome Atlas; TFAP2A, transcription factor AP-2 (activating enhancer binding protein 2) alpha; TPM, transcripts per kilobase million.

To evaluate the clinical significance of TFAP2A, survival analysis was performed using the Kaplan-Meier Plotter database. NSCLC patients with high TFAP2A expression had significantly shorter overall survival times compared to those with low expression levels (Figure 1C; HR =1.64, 95% CI: 1.30–2.06; P<0.001). These findings suggest that TFAP2A upregulation is associated with poor prognosis in NSCLC patients.

Further, we explored the relationship between TFAP2A expression and cisplatin resistance in NSCLC cells. RT-qPCR revealed that TFAP2A mRNA levels were significantly increased in cisplatin-resistant NSCLC cells (A549-Re and PC9-Re) compared to their cisplatin-sensitive counterparts (Figure 1D). Consistent with the mRNA data, western blot analysis demonstrated elevated TFAP2A protein expression in the cisplatin-resistant NSCLC cells (Figure 1E). Together, these results highlighted that TFAP2A was overexpressed in NSCLC and was closely linked to cisplatin resistance and poor patient survival, suggesting a potential role for TFAP2A in drug resistance mechanisms in NSCLC.

TFAP2A intervention enhances cisplatin-sensitivity in NSCLC cells

In order to explore the impact of TFAP2A silencing on cisplatin-resistant NSCLC cells, si-TFAP2A was constructed and confirmed to significantly downregulate TFAP2A expression at both mRNA and protein levels in A549-Re and PC9-Re cells compared to the si-Control group (Figure 2A,2B). Based on the validation results from PCR and western blot assays, si-TFAP2A-2 (5'-CCCAGATCAAACTGTAATTAA-3') was ultimately selected for subsequent experimental investigations.

Figure 2.

Silencing of TFAP2A inhibits proliferation in cisplatin-resistant NSCLC cells. (A,B) RT-qPCR and western blot assays were applied to detect the level of TFAP2A mRNA (A) and protein (B) in cisplatin-resistant NSCLC cells; (C) CCK-8 assay was used to evaluate proliferation of cisplatin-resistant NSCLC cells under cisplatin treatment; (D) clone formation assay (crystal violet staining) was performed to assess proliferation of cisplatin-resistant or sensitive NSCLC cells; (E) PI fluorescence assay was conducted to detect apoptosis in cisplatin-resistant NSCLC cells. (F) MitoSOX assays was utilized to detect apoptosis in cisplatin-resistant NSCLC cells by flow cytometry. Data were reported as mean and SD. CCK-8, cell counting kit-8; NSCLC, non-small cell lung cancer; PI, propidium iodide; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TFAP2A, transcription factor AP-2 (activating enhancer binding protein 2) alpha.

To assess the impact of TFAP2A knockdown on cisplatin sensitivity, a CCK-8 assay was performed. The results revealed that silencing TFAP2A markedly enhanced the sensitivity of A549-Re and PC9-Re cells to increasing concentrations of cisplatin, as indicated by a significant reduction in cell viability compared to si-Control cells (Figure 2C).

Furthermore, clone formation assays demonstrated that TFAP2A silencing effectively suppressed cell proliferation in both cisplatin-resistant (A549-Re and PC9-Re) and cisplatin-sensitive NSCLC cells (A549-Se and PC9-Se) (Figure 2D).

To explore the role of TFAP2A in apoptosis, a PI fluorescence assay was performed. The results showed a substantial increase in apoptosis following TFAP2A knockdown in A549-Re and PC9-Re cells, as evidenced by enhanced PI staining (Figure 2E). Similarly, apoptosis was further validated via Annexin V-FITC/PI dual-staining assay (Figure S1), corroborating the aforementioned results.

Additionally, MitoSOX assays were used to detect mitochondrial oxidative stress, which is a key marker of apoptosis. TFAP2A silencing resulted in a significant increase in mitochondrial ROS levels, further confirming its role in promoting apoptosis in cisplatin-resistant NSCLC cells (Figure 2F). These results suggested that targeting TFAP2A could be a promising strategy for overcoming cisplatin resistance in NSCLC.

TFAP2A transcriptionally regulates BNIP3 expression and promotes mitophagy in cisplatin-resistant NSCLC cells

Typically, NSCLC cells selectively encapsulate and degrade mitochondria through autophagy mechanisms to maintain homeostasis (24). BNIP3, a multifunctional protein located on the outer mitochondrial membrane, can function as either an apoptotic-inducing protein or a promoter of mitophagy (25,26). To evaluate its role in cisplatin-resistant NSCLC cells, we first analyzed the mRNA levels and transcriptional activity of BNIP3. RT-qPCR analysis showed that BNIP3 mRNA expression was significantly higher in cisplatin-resistant A549-Re and PC9-Re cells compared to their cisplatin-sensitive counterparts (Figure 3A). Consistently, a dual-luciferase reporter assay demonstrated elevated BNIP3 promoter activity in resistant cells (Figure 3B), suggesting enhanced BNIP3 transcription in cisplatin-resistant NSCLC cells.

Figure 3.

TFAP2A transcriptionally regulates BNIP3 expression and promotes mitophagy in cisplatin-resistant NSCLC cells. (A) RT-qPCR analysis of BNIP3 mRNA levels in NSCLC cells; (B) dual-luciferase reporter assay was applied to assess BNIP3 transcriptional activity in NSCLC cells; (C) Western blot analysis of autophagy-related proteins LC3I, LC3II, p62, COX IV, TOMM20, and BNIP3 expression levels in NSCLC cells; (D) JASPAR prediction of TFAP2A binding sites on the BNIP3 promoter; (E) dual-luciferase reporter assay was used to detect TFAP2A transcriptional activity in cisplatin-resistant NSCLC cells; (F) RT-qPCR analysis of BNIP3 mRNA levels in carboplatin-resistant NSCLC cells; (G) ICC was performed to detect MitoSOX fluorescence expression in cisplatin-resistant NSCLC cells; (H) Western blot analysis of autophagy-related proteins LC3I, LC3II, p62, COX IV, TOMM20, and BNIP3 expression levels in cisplatin-resistant NSCLC cells with or absent of si-TFAP2A; (I) immunofluorescence was conducted to detect LC3B expression levels in cisplatin-resistant NSCLC cells. Data were reported as mean and SD. A P value less than 0.05 was considered to indicate a statistically significant difference. BNIP3, BCL2/adenovirus ElB 19kDa interacting protein 3; COX IV, cytochrome c oxidase subunit IV; JASPAR, Journal of American Society of Phlebotomy Technicians; LC3B, microtubule associated protein 1 light chain 3 beta; LC3I, microtubule-associated protein 1A/1B-light chain 3-I; LC3II, microtubule-associated protein 1A/1B-light chain 3-II; NSCLC, non-small cell lung cancer; p62, sequestosome 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TFAP2A, transcription factor AP-2 (activating enhancer binding protein 2) alpha; TOMM20, translocase of outer mitochondrial membrane 20.

Next, we assessed the levels of mitophagy in these cells. Western blot analysis revealed increased levels of LC3-II, a hallmark of autophagy activation, and decreased levels of p62 in cisplatin-resistant cells compared to sensitive cells. Additionally, the mitochondrial proteins COX IV and TOMM20 were downregulated, while BNIP3 protein expression was elevated, confirming enhanced mitophagy in cisplatin-resistant NSCLC cells (Figure 3C).

To determine the regulatory relationship between TFAP2A and BNIP3, we used JASPAR bioinformatics predictions (https://jaspar.elixir.no/), which identified putative TFAP2A binding sites on the BNIP3 promoter (Figure 3D). To validate this, we performed site-directed mutagenesis of the BNIP3 promoter. In a dual-luciferase assay, TFAP2A binding significantly enriched BNIP3 transcriptional activity, whereas mutating the predicted binding sites abolished this effect (Figure 3E).

To further confirm the regulatory role of TFAP2A in BNIP3 expression, we silenced TFAP2A in cisplatin-resistant NSCLC cells. RT-qPCR analysis demonstrated a significant reduction in BNIP3 mRNA levels in si-TFAP2A-transfected A549-Re and PC9-Re cells compared to the si-Control group (Figure 3F). Concurrently, MitoSOX fluorescence assays showed a marked accumulation of mitochondrial ROS in si-TFAP2A-treated cells, suggesting impaired mitophagy and increased mitochondrial stress (Figure 3G).

Additionally, Western blot analysis of mitophagy-related proteins revealed that TFAP2A knockdown led to a reduction in LC3-II and an accumulation of p62, along with increased COX IV and TOMM20 protein levels, indicating significant inhibition of mitophagy (Figure 3H).

Lastly, immunofluorescence analysis of LC3B expression further confirmed reduced mitophagy in si-TFAP2A-treated A549-Re and PC9-Re cells (Figure 3I).

In summary, these results demonstrated that TFAP2A transcriptionally regulated BNIP3, promoting mitophagy in cisplatin-resistant NSCLC cells. Knockdown of TFAP2A suppressed BNIP3 expression, impaired mitophagy, and increased mitochondrial ROS accumulation, providing insight into TFAP2A’s role in cisplatin resistance.

TFAP2A intervention attenuates NSCLC cell proliferation by modulating mitophagy in cisplatin-resistant cells

To determine whether TFAP2A promotes proliferation by modulating mitophagy, we assessed the effects of RAPA in cisplatin-resistant NSCLC cells. Cell proliferation assays showed that RAPA significantly enhanced the proliferation of A549 and PC9 cells, while TFAP2A silencing suppressed this RAPA-induced proliferation (Figure 4A). Similarly, RAPA-induced inhibition of apoptosis was reversed upon TFAP2A knockdown, as evidenced by increased apoptosis and mitochondrial ROS levels (Figure 4B-4D). These results indicated that TFAP2A intervention disrupted the protective effects of mitophagy on cellular homeostasis.

Figure 4.

TFAP2A modulates cell proliferation via mitophagy in cisplatin-resistant NSCLC. (A) Assessment of cisplatin-resistant NSCLC cells proliferation via cell cloning experiments (crystal violet staining); (B) detection of cell apoptosis through PI staining; (C,D) measurement of mitochondrial ROS levels in cisplatin-resistant NSCLC cells using MitoSOX staining; (D) flow cytometry to detect mitochondrial ROS levels in cisplatin-resistant NSCLC cells; (E) Western blot analysis of autophagy-related proteins LC3I, LC3II, p62, COX IV, TOMM20, and BNIP3 expression levels in cisplatin-resistant NSCLC cells with or absent of si-TFAP2A; (F) immunofluorescence to detect LC3B expression levels in cisplatin-resistant NSCLC cells. Data were reported as mean and SD. BNIP3, BCL2/adenovirus ElB 19kDa interacting protein 3; COX IV, cytochrome c oxidase subunit IV; DAPI, 4’,6-diamidino-2-phenylindole; LC3B, microtubule associated protein 1 light chain 3 beta; LC3I, microtubule-associated protein 1A/1B-light chain 3-I; LC3II, microtubule-associated protein 1A/1B-light chain 3-II; NSCLC, non-small cell lung cancer; p62, sequestosome 1; PI, propidium iodide; RAPA, rapamycin; ROS, reactive oxygen species; SD, standard deviation; TFAP2A, transcription factor AP-2 (activating enhancer binding protein 2) alpha; TOMM20, translocase of outer mitochondrial membrane 20.

Western blot analysis revealed that RAPA increased LC3-II expression and decreased p62 levels, consistent with enhanced mitophagy. However, TFAP2A silencing significantly reduced LC3-II levels, restored p62, and increased mitochondrial protein expression (COX IV and TOMM20), indicating impaired mitophagy (Figure 4E). Additionally, BNIP3 protein levels, a key mitophagy regulator, were significantly downregulated after TFAP2A intervention. Immunofluorescence analysis confirmed these findings, showing reduced colocalization of LC3B with mitochondria and diminished LC3B puncta following TFAP2A knockdown, despite RAPA treatment (Figure 4F).

In brief, TFAP2A enhanced BNIP3-mediated mitophagy to promote proliferation and suppress apoptosis in cisplatin-resistant NSCLC cells. TFAP2A silencing disrupted mitophagy, leading to suppressed proliferation, increased apoptosis, and elevated mitochondrial ROS levels.

TFAP2A promotes NSCLC cell mitophagy and tumor growth in xenograft nude mouse model

To evaluate the role of TFAP2A in regulating mitophagy and tumor growth in vivo, we established a xenograft nude mouse model with cisplatin-resistant A549 NSCLC cells. After a monitoring period of 28 days, tumor progression was assessed. The findings indicated that both the volume and the growth rates of tumors were markedly suppressed in the sh-TFAP2A group when contrasted with the sh-Control group. Notably, treatment with RAPA partially reversed this inhibitory effect, promoting tumor growth in the sh-TFAP2A group (Figure 5A).

Figure 5.

TFAP2A promotes NSCLC mitophagy and tumor growth in xenograft tumor model of nude mice. (A) Photographs of tumors on day 28 and changes in tumor volume on days 8, 13, 18, 23, and 28 in nude mice with transplanted cisplatin-resistant A549 cells; (B) RT-qPCR analysis of TFAP2A mRNA levels in tumor tissues; (C) HE staining was used to assess inflammatory infiltration in xenograft tumor model of nude mice; (D) TUNEL staining was performed to detect apoptosis in xenograft tumor model of nude mice; (E) Ki-67 immunohistochemical staining was applied to evaluate cell proliferation in xenograft tumor model of nude mice; (F,G) immunohistochemistry analysis of TFAP2A (F) and BNIP3 (G) expression levels in xenograft tumor model of nude mice. Data were reported as mean and SD. A P value less than 0.05 was considered to indicate a statistically significant difference. BNIP3, BCL2/adenovirus ElB 19kDa interacting protein 3; HE, hematoxylin and eosin; Ki-67, antigen Kiel 67; NSCLC, non-small cell lung cancer; RAPA, rapamycin; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TFAP2A, transcription factor AP-2 (activating enhancer binding protein 2) alpha; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

RT-qPCR analysis of tumor tissues confirmed that TFAP2A mRNA levels were significantly reduced in the sh-TFAP2A group, verifying the effectiveness of TFAP2A knockdown (Figure 5B).

To further investigate the tumor microenvironment and cellular changes, HE staining was performed. Tumors from the sh-TFAP2A group showed reduced inflammatory cell infiltration compared to the sh-Control group, while RAPA treatment restored inflammatory infiltration (Figure 5C). TUNEL staining revealed that apoptosis levels were significantly increased in the sh-TFAP2A group, further supporting the inhibitory effects of TFAP2A knockdown on tumor growth. In contrast, RAPA intervention reduced apoptosis in sh-TFAP2A tumors (Figure 5D).

To assess cell proliferation, Ki67 immunohistochemical staining was conducted. Tumors in the sh-TFAP2A group exhibited a marked reduction in Ki67-positive cells, indicating suppressed proliferation. However, RAPA treatment significantly increased Ki67 expression, demonstrating its ability to promote cell proliferation even in the absence of TFAP2A (Figure 5E).

Finally, immunohistochemistry analysis of TFAP2A and BNIP3 expression levels in tumor tissues further confirmed the in vivo findings. TFAP2A expression was significantly reduced in the sh-TFAP2A group, accompanied by a downregulation of BNIP3 protein levels, supporting the role of TFAP2A in BNIP3-mediated mitophagy. RAPA treatment partially restored BNIP3 expression, correlating with the observed tumor growth promotion (Figure 5F,5G).

In summary, these results demonstrated that TFAP2A knockdown suppressed tumor growth in vivo by reducing cell proliferation, inflammatory infiltration, and BNIP3-mediated mitophagy while enhancing apoptosis. RAPA treatment could partially rescue these effects, further highlighting the role of TFAP2A in modulating mitophagy and tumor progression in NSCLC.

Discussion

This research aimed to explore the function of TFAP2A in enhancing cell proliferation in NSCLC through its regulatory effect on mitophagy. The results indicated that high expression of TFAP2A was associated with cisplatin-resistant in NSCLC patients and NSCLC cells. Knockdown of TFAP2A significantly enhanced the sensitivity of NSCLC cells to cisplatin, inhibited their proliferation, and promoted cell apoptosis. Notably, TFAP2A regulates the expression of BNIP3 and mitophagy. In NSCLC cells, mutation of the binding sites between TFAP2A and the promoter region of BNIP3 significantly inhibited the transcriptional activity of BNIP3. Furthermore, the knockdown of TFAP2A led to the accumulation of mitochondrial ROS and inhibition of mitophagy in NSCLC cells. Additional in vivo studies demonstrated that knockdown of TFAP2A significantly inhibited the growth of NSCLC transplanted tumors, reduced the expression of Ki67, and increased cell apoptosis.

Firstly, we conducted database exploration and in vitro studies to demonstrate the negative correlation of TFAP2A with NSCLC patients and cellular cisplatin resistance. Indeed, increasing data has suggested that the silencing of TFAP2A could inhibit the proliferation of cisplatin-resistant NSCLC cells. Conversely, the literature indicated that DACT2, regulated transcriptionally by TFAP2A-mediated allelic variation, could enhance the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced LUAD (27). However, an increasing amount of data indicated that the silencing of TFAP2A inhibited the proliferation of drug-resistant NSCLC cells (28). For instance, in anlotinib-resistant cells, TFAP2A was upregulated and associated with angiogenesis-related processes. Knocking out TFAP2A significantly reduced tumor-induced angiogenesis and partially rescued the anti-angiogenic activity of anlotinib (13). Consistent with these findings, our study also confirmed a positive correlation between TFAP2A and tumor proliferation, as tumor growth was inhibited following the knockout of TFAP2A. The results indicate that TFAP2A may serve as a promising therapeutic target to address cisplatin resistance in NSCLC.

In addition to its role in cisplatin resistance, our study also highlighted the regulatory effect of TFAP2A on mitophagy in cisplatin-resistant NSCLC cells. Mitophagy is a crucial cellular process involving the selective degradation and recycling of damaged mitochondria (29). LC3 serves as a pivotal marker in this process, with the conversion from LC3I to LC3II signifying the formation of autophagosomes (30,31). p62, a shuttle protein, binds to ubiquitinated proteins and subsequently to LC3II, forming complexes that undergo degradation within lysosomes (32,33). COXIV is an enzyme complex located on the mitochondrial inner membrane, and is integral to the mitochondrial respiratory chain (34,35). TOMM20, a subunit of the mitochondrial outer membrane translocase, facilitates the entry of proteins into mitochondria (36). It is also well-known that abnormalities in mitochondria directly lead to abnormal cell death (37). Furthermore, functional impairments in COX IV and TOMM20 may affect mitochondrial respiratory function and protein translocation, ultimately influencing tumor cells energy metabolism and survival (38). BNIP3, one of the receptors for mitophagy, directly binds to LC3, promoting the formation of autophagosomes (39,40). Our findings suggested that TFAP2A could regulate the expression of BNIP3, a regulatory relationship that was not identified before. In cisplatin-resistant NSCLC cells, silencing TFAP2A led to reducing BNIP3 expression, decreasing mitophagy. These findings were also supported by recent literature, which indicated that BNIP3 plays a crucial role in regulating mitophagy and that its expression was often altered in drug-resistant cancer cells (28).

In a pivotal discovery, our study also revealed that variations in BNIP3 led to decreased abundance of TFAP2A, hinting at the existence of binding sites between TFAP2A and BNIP3. Our research revealed that mitophagy regulated by TFAP2A significantly increased the resistance of NSCLC cells to apoptosis, as observed in both in vitro and in vivo studies. We confirmed that the suppression of TFAP2A-mediated mitophagy rendered tumor cells more susceptible to apoptosis, indicating that mitophagy serves a crucial role in safeguarding and promoting the survival of these cancer cells. When we utilized si-TFAP2A, a significant increase in apoptosis and a substantial inhibition of proliferation were observed in NSCLC cells. Conversely, applying the mitophagy agonist to NSCLC cells treated with si-TFAP2A significantly ameliorated the inhibition of apoptosis, providing evidence that TFAP2A protected cells by activating BNIP3-mediated mitochondrial fission and mitophagy. Notably, based on our current research findings, TFAP2A was significantly upregulated in cisplatin-resistant NSCLC. It could modulate the cisplatin resistance of NSCLC following intervention, thereby emerging as a potential therapeutic target for overcoming chemotherapy resistance in NSCLC.

This study found that sh-TFAP2A-resistant NSCLC grew more slowly in vivo with relatively smaller tumor tissue. However, with additional RAPA intervention, tumor tissue appeared to increase significantly, reflecting that TFAP2A acts on mitophagy to promote tumor tissue growth. Similarly, BNIP3 was found to show consistent results with TFAP2A expression in immunohistochemistry experiments, also suggesting the possibility of its targeted regulation. Moreover, based on the mechanism pathways identified in this study, the therapeutic potential of small molecules should be explored in future research. To our knowledge, there are currently no validated specific inhibitors of TFAP2A and BNIP3 with conclusive in vivo efficacy. This might be a significant flaw in translational research, and efforts should be made to develop pathway-specific agents in the future.

Conclusions

In summary, we identified TFAP2A as a key player in promoting cell proliferation and drug resistance in NSCLC by regulating BNIP3-mediated mitophagy, suggesting its potential as a therapeutic target to overcome chemotherapy resistance in NSCLC.

Supplementary

The article’s supplementary files as

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. All animal experiments were performed under a project license (No. KS24001) granted by the Ethics Committee of Shanghai Chest Hospital, in compliance with China national or institutional guidelines for the care and use of animals.

Data Sharing Statement

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DOI: 10.21037/tcr-2025-646